Four-factor prothrombin complex concentrate reverses apixaban-associated bleeding in a rabbit model of acute hemorrhage.

BACKGROUND: Apixaban is a direct factor Xa inhibitor approved for the treatment and prevention of thromboembolic disease. There is a lack of data regarding its reversal in cases of acute bleeding or prior to emergency surgery that needs addressing. OBJECTIVES: This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®) /Kcentra(®) , CSL Behring) can effectively reverse apixaban-associated bleeding in an in vivo rabbit model and evaluated the correlations between in vivo hemostasis and in vitro coagulation parameters. METHODS: For dose-finding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800-1600 µg kg(-1) ) and, following a standardized kidney incision, volume of blood loss and time to hemostasis were measured. In a subsequent study phase, anesthetized rabbits were treated with apixaban 1200 µg kg(-1) followed by 4F-PCC (6.25-100 IU kg(-1) ), and the effects on the same bleeding parameters were assessed. In parallel, coagulation parameters were monitored. RESULTS: Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration. Preincision treatment with 4F-PCC resulted in a statistically significant reversal in bleeding time (all doses) and volume (doses ≥ 12.5 IU kg(-1) ). Of the coagulation parameters measured, thrombin generation initiated using the RD reagent (phospholipids only) was the most sensitive to in vivo measures of 4F-PCC's hemostatic efficacy, although some correlations were also observed for prothrombin time and whole blood clotting time. CONCLUSIONS: In this rabbit model of acute hemorrhage, 4F-PCC showed potential for reversing the bleeding effects of apixaban. Clinical data in apixaban-treated patients are needed to confirm these results.

Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP).

BACKGROUND: Recombinant factor VIIa (rFVIIa) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy. Due to its short half-life (t½), frequent injections are required, limiting its use as a prophylactic treatment. A novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) has been developed to extend the t(½) of rFVIIa. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic/pharmacodynamic characteristics of rVIIa-FP in preclinical animal species. METHODS: Pharmacokinetic (PK) parameters were derived after single intravenous dosing in hemophilia A mice, rats, rabbits and monkeys. PK analysis was based on human FVII plasma levels determined by measuring FVII antigen levels by ELISA in mice and rats, and FVIIa activity using STACLOT® VIIa-rTF in rabbits and monkeys. Induction of thrombin generation was investigated in mice, while hemostatic activity was assessed by thrombus formation in rabbits. RESULTS: Compared with rFVIIa, rVIIa-FP displayed a prolonged t(½), enhanced in vivo recovery and reduced clearance in all species investigated. In mice, 16 h after treatment with rVIIa-FP, thrombin levels were quantifiable, indicating prolonged efficacy, whereas values had approached baseline at this time after treatment with rFVIIa. After 12 h, hemostatic efficacy was negligible in rFVIIa-treated rabbits, but sustained in animals receiving rVIIa-FP. CONCLUSIONS: These studies indicate that the longer t(½) of rVIIa-FP compared with rFVIIa translates into extended activity. These findings suggest that rVIIa-FP has the potential to be administered less frequently than rFVIIa-containing concentrates in clinical use.

Reversal of dabigatran anticoagulation by prothrombin complex concentrate (Beriplex P/N) in a rabbit model.

BACKGROUND: One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation. OBJECTIVES: This study was designed to determine in an animal model whether a PCC (Beriplex P/N) can effectively reverse the effects of dabigatran. An additional objective was to evaluate markers of dabigatran-associated bleeding diathesis. METHODS: Anesthetized rabbits were treated with 0.4 mg kg(-1) dabigatran followed by PCC doses of 20, 35 or 50 IU kg(-1) or placebo. After a standardized kidney incision, volume of blood loss and time to hemostasis were determined. RESULTS: From an initial mean of 29 mL, blood loss progressively declined by 5.44 mL with a 95% confidence interval (CI) of 2.21-8.67 mL per 10 IU kg(-1) increment in PCC dose (P = 0.002). At a PCC dose of 50 IU kg(-1) blood loss was fully normalized. Increasing PCC doses shortened the median time to hemostasis from 20.0 to 5.7 min (P < 0.001). The rate of hemostasis was nearly trebled with each 10 IU kg(-1) increment in PCC dose (rate ratio, 2.89; CI, 1.64-5.09). CONCLUSIONS: In this animal study, PCC showed potential as an agent for reversing the effects of dabigatran. Further investigation is warranted.

Improved kinetics of rIX-FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs.

BACKGROUND: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). METHODS: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. RESULTS: The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). CONCLUSIONS: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.

Repeated infusions of VWF/FVIII concentrate: impact of VWF:FVIII ratio on FVIII trough and peak levels in a rabbit model.

The ratio of von Willebrand factor (VWF) to FVIII differs among available VWF/FVIII concentrates. Repeated infusions of concentrates with a low VWF:FVIII ratio may expose patients with von Willebrand disease to supranormal FVIII levels. The aim of this study was to determine the effects of repeated infusions with two VWF/FVIII concentrates differing in VWF:FVIII ratio on attained FVIII trough and peak levels as well as other pharmacokinetic parameters. Rabbits were randomized to receive multiple 150 IU kg⁻¹ VWF:RCo infusions at 4 h intervals with VWF/FVIII concentrates of a high (Haemate® P/Humate-P®) or low (Wilate®) VWF:FVIII ratio. Trough plasma FVIII and VWF levels were measured after each infusion. Pharmacokinetic analysis was performed using samples collected frequently after infusion. Mean FVIII trough level after the first Wilate infusion was 50.6 IU dL⁻¹ with a 95% confidence interval (CI) of 43.1-58.2 IU dL⁻¹, compared with 31.8 IU dL⁻¹ (CI, 24.4-39.1 IU dL⁻¹) for Haemate P (P<0.001). Trough levels progressively increased over the 24 h treatment period in both groups. After the final infusion, mean trough FVIII remained significantly higher (P = 0.002) in recipients of Wilate. Mean peak FVIII concentration after infusion was 67% higher in the Wilate group (167 vs. 100 IU dL⁻¹ , respectively; P = 0.002). Mean cumulative exposure to FVIII, as measured by area under the curve, was 84% greater in Wilate-treated animals. Half-life did not differ between the two concentrates. Animal model data suggest that exposure to elevated FVIII levels can be reduced through use of VWF/FVIII concentrates with higher VWF:FVIII ratios.

Prothrombin complex concentrate mitigates diffuse bleeding after cardiopulmonary bypass in a porcine model.

BACKGROUND: Extracorporeal circuit priming and intravascular volume expansion during cardiopulmonary bypass (CPB) may lead to dilutional coagulopathy and excessive diffuse postoperative bleeding. Prothrombin complex concentrate (PCC) containing clotting factors II (FII), VII (FVII), IX (FIX), and X (FX) could be of potential value in correcting dilutional coagulopathy and reducing blood loss. METHODS: Anaesthetized pigs underwent CPB with hypothermia for 2 h at 25°C followed by 1 h of normothermia. Approximately 1 h after CPB, animals randomly received either isotonic saline 1 ml kg⁻¹ or PCC 30 IU kg⁻¹ in a volume of 1 ml kg⁻¹. Diffuse coagulopathic bleeding was assessed as suture hole blood loss from a Gore-Tex patch placed over a full-thickness incision in the left carotid artery. RESULTS: After CPB, levels of FII, FVII, FIX, and FX declined from baseline by 32% to 48%, and PCC fully or partially reversed those deficits. Median suture hole blood loss after administration of saline placebo was 74 ml. PCC reduced suture hole bleeding by a median of 54 ml with a 95% confidence interval of 6-112 ml (P=0.026) compared with saline. PCC, but not saline, normalized skin bleeding time. Peak thrombin generation markedly decreased after CPB, but then returned in PCC-treated animals to a level higher than baseline by 28.7 nM (14.5-41.1 nM; P=0.031). CONCLUSIONS: PCC was effective in correcting dilutional coagulopathy and reducing diffuse bleeding in an in vivo large-animal CPB model. Further research is warranted on PCC as a haemostatic agent in CPB.

Prothrombin complex concentrate vs fresh frozen plasma for reversal of dilutional coagulopathy in a porcine trauma model.

BACKGROUND: Fluid resuscitation following traumatic injury causes haemodilution and can contribute to coagulopathy. Coagulation factor replacement may be necessary to prevent bleeding complications of dilutional coagulopathy. Compared with fresh frozen plasma (FFP), prothrombin complex concentrate (PCC) may potentially offer a more rapid and effective means of normalizing coagulation factor levels. METHODS: In anaesthetized mildly hypothermic pigs, 65-70% of total blood volume was substituted in phases with hydroxyethyl starch and red cells. Animals were then treated with 15 ml kg(-1) isotonic saline placebo, 25 IU kg(-1) PCC, or 15 ml kg(-1) FFP. Immediately thereafter, either a standardized femur or spleen injury was inflicted, and coagulation function, including thrombin generation, and bleeding were assessed. An additional group received high-dose FFP (40 ml kg(-1)) before femur injury. RESULTS: Haemodilution markedly prolonged prothrombin time and reduced peak thrombin generation. PCC, but not FFP, fully reversed those effects. Compared with 15 ml kg(-1) FFP, PCC shortened the time to haemostasis after either bone (P=0.001) or spleen (P=0.028) trauma and reduced the volume of blood lost (P<0.001 and P=0.015, respectively). Subsequent to bone injury, PCC also accelerated haemostasis (P=0.003) and diminished blood loss (P=0.006) vs 40 ml kg(-1) FFP. CONCLUSIONS: PCC was effective in correcting dilutional coagulopathy and controlling bleeding in an in vivo large-animal trauma model. In light of its suitability for more rapid administration than FFP, PCC merits further investigation as a therapy for dilutional coagulopathy in trauma and surgery.

Prevention of gynaecological adhesions using haemostatic fleece in a rabbit model.

This pre-clinical study was performed to investigate the ability of the haemostatic fleece TachoComb to prevent adhesion formation following uterine surgery. Thirty rabbits were randomized to receive TachoComb or no intervention following incision to the right uterine horn. After 14 days, the animals were killed and examined for the presence of adhesion. The lengths of any adhesions were measured and the severity was recorded as a score (0, no adhesion; 1, adhesion easy to lyse; 2, adhesion lysed with traction; 3, adhesion separated by sharp dissection). The incidence of adhesions was 100% in the control group compared with 33% in the TachoComb-treated animals. The mean adhesion score was significantly lower (0.7 versus 2.2) and the mean adhesion length category was significantly shorter (0.4 versus 2.0) with TachoComb than in the control group. This study indicates that TachoComb is a well-tolerated and effective means of preventing adhesion following gynaecological surgery.

Fibrin sealants in supporting surgical techniques: strength in factor XIII.

Factor XIII has a well-established role in natural coagulation and clot stabilization. It is often added back to fibrin sealants that are used in a wide range of surgical settings to achieve successful hemostasis, tissue adhesion and wound healing. Factor XIII is the final enzyme to be activated in the blood coagulation cascade. It plays an important role in maintaining the balance between coagulation and fibrinolysis. Factor XIII facilitates the formation of covalent cross-links within the fibrin network, forming a loose mesh after activation by thrombin. It adds significant resilience to fibrin clots, augmenting strength by as much as 5-fold. Both fibrin cross-linking and the factor XIII-catalyzed ligation of the fibrinolysis inhibitor alpha(2)-antiplasmin to the fibrin clot contribute to the increased proteolytic resistance of factor XIII-stabilized clots. Preclinical studies indicate that the inclusion of factor XIII in fibrin sealants used for vascular grafting significantly reduces suture-hole blood loss. This has important implications for the successful control of bleeding in comparable clinical situations. The advantages of factor XIII stabilized clots (increased strength, resistance to proteolysis, promotion of wound healing) suggest that the presence of factor XIII in fibrin sealants may optimize their performance in the clinical setting. The aim of this paper is to review preclinical data that provide evidence for a potentially positive role for factor XIII in fibrin sealants.

Treatment of porcine sepsis with high-dose antithrombin III reduces tissue edema and effusion but does not increase risk for bleeding.

We evaluated the effectiveness of antithrombin III (AT III) infusions designed to achieve supraphysiologic plasma levels of this serine protease inhibitor in preventing vascular permeability and disseminated intravascular coagulation in a pig model of sepsis. In addition, we determined whether high AT III doses were associated with increased bleeding risk. Sepsis was induced in 18 pigs by injection of lipopolysaccharide (LPS) (0.25 microg/kg per h for 3 h). At 90 min after the start of LPS infusion, pigs were randomized (n = 6 per group) to receive either human serum albumin as a placebo, AT III 120/5 (120 U/kg, 30-min bolus + 5 U/kg per h for 240 min), or AT III 250/10 (250 U/kg + 10 U/kg per h). Three additional animals served as negative controls (no LPS, no AT III). Treatment with AT III significantly reduced the amount of effluents in body cavities and fibrin monomers. AT III did not significantly increase bleeding risk as determined by organ hemorrhage. An additional assessment of AT III's bleeding risk [skin bleeding time (SBT)] was carried out in 35 nonseptic pigs treated with either AT III alone (120/5 or 250/10) or in the combination with heparin. Heparin administration alone produced a dose-dependent increase in SBT, but AT III alone did not. Addition of AT III 120/5 to heparin did not induce a further increase in bleeding time over heparin alone. These results indicate that administration of AT III in doses designed to achieve very high plasma concentrations significantly ameliorates symptoms of sepsis-induced vascular leakage and disseminated intravascular coagulation without increasing bleeding risk.

The potentiation of human C1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model.

C1-inhibitor (C1-Inh) is an important regulator of inflammatory reactions because it is a potent inhibitor of the contact and complement system. C1-Inh application in inflammatory disease is, however, restricted because of the high doses required. The glycosaminoglycan-like molecule dextran sulphate (DXS) enhances C1-Inh function in vitro. Hence, we investigated whether co-administration with dextran sulphate reduces the amount of C1-Inh required, through enhancement in vivo. C1-Inh potentiation was measured in a newly developed C1s-inactivation assay that is based on activation of C4 by purified C1s. Activated C4 in rat plasma was quantified with a newly developed ELISA. Human C1-Inh (2.5 microM) inhibited C1s in rat plasma 55-fold faster in the presence of dextran sulphate (15 kDa, 5 microM). To study the stability of the complex in vivo, rats were given a mixture of C1-Inh (10 mg/kg) and dextran sulphate (3 mg/kg). C1-Inh activity during 5 h was analyzed ex vivo with the C1s inactivation assay. The noncovalent C1-Inh-dextran sulphate complex resulted in a transient enhancement of the inhibitory capacity of C1-Inh, lasting for 60-90 min. Dextran sulphate did not affect plasma clearance of C1-Inh. We conclude that the enhanced inhibitory capacity of C1-Inh complexed to dextran sulphate is transient in vivo. Hence, co-administration of these compounds seems a feasible approach to achieve short-term inhibition of complement in vivo.

Prevention of suture hole bleeding using fibrin sealant: benefits of factor XIII.

BACKGROUND: Suture hole bleeding is a frequent complication in vascular surgery. The use of fibrin sealants (FSs) during surgery is reported to reduce blood loss by enhancing hemostasis. OBJECTIVES: Using a pig vascular graft model we investigated: (1) the use of FSs in vascular surgery and measured blood loss with and without the use of FSs; (2) the effect of factor XIII in FSs on hemostasis and blood loss; and (3) the effect of increasing FS incubation time on hemostasis and blood loss. MATERIALS AND METHODS: During surgery, a 3-cm incision was made in the carotid artery wall. Incisions were covered with a Gore-Tex (PTFE) patch sutured with Ethicon monofil 5/0 or 3/0. The resulting suture holes were treated according to study protocol. Study 1: Nine pigs received either FS treatment (n = 5) or no treatment (n = 4). Study 2: Twenty pigs underwent bilateral surgery; right (n = 20) and left (n = 20) patches received either FS with factor XIII (FS + FXIII) or FS depleted of factor XIII (FS - FXIII). Study 3: Bilateral surgery was carried out on 24 pigs; PFTE patches secured with Ethicon 3/0 were treated with FS and allowed to polymerize for 120 s (n = 12), 180 s (n = 12), or 240 s (n = 12) or with solid support/thrombin (n = 12). RESULTS: Study 1: Mean blood loss was significantly lower in the FS-treated group compared with untreated controls (9.2 +/- 10.6 mL vs 178.8 +/- 125.5 mL, mean +/- SD, P = 0.016). Study 2: Significantly less blood was lost in the FS + FXIII group than in the FS - FXIII group (Delta = 9.6 mL, P = 0. 02). Study 3: Blood loss was significantly higher from patches treated with solid support/thrombin compared with those treated with FS (P < 0.01). CONCLUSIONS: FSs containing factor XIII, such as Beriplast P, are effective in reducing suture hole bleeding and improving hemostasis during vascular graft procedures. The presence of factor XIII contributes to the efficacy of FSs and reduces the time to hemostasis. During vascular surgery, blood loss can be reduced significantly by the use of fibrin sealant compared with absorbable gelatin sponges coated with thrombin.

Influence of antithrombin III on coagulation and inflammation in porcine septic shock.

The physiological inhibitor of thrombin, antithrombin III (ATIII, Kybernin P) was investigated for its antiinflammatory and anticoagulant effects in a pig model of septic shock. Pigs were infused with a dose of 0.25 microgram. kg-1. h-1 of lipopolysaccharide (LPS) over a period of 3 hours. Animals developed systemic inflammation, disseminated intravascular coagulation (DIC), organ failure and cardiovascular abnormalities, namely pulmonary hypertension and systemic hypotension. Twenty septic pigs were allocated to 2 study groups, treated either with ATIII (n=10) or placebo (n=10). ATIII was administered as a 250-U/kg IV bolus infusion for 30 minutes (-60 to -30 minutes) followed by a single IV bolus of 125 U/kg (t=0) and a second 30-minute infusion of 250 U/kg (120 to 150 minutes). ATIII significantly prevented the development of a DIC; the increase in fibrin monomers (placebo, 11.4+/-9.1 reciprocal titers, at 6 hours) was completely overcome by ATIII (P<0. 05). ATIII significantly prevented the increase in thromboxane (TXB2) levels, which were 809+/-287 pg/mL in the placebo and 420+/-174 pg/mL in the verum group after 6 hours (P<0.02). On the other hand, ATIII had no influence on TNF levels. In a lethal study with an increased dose of LPS (0.5 microgram. kg-1. h-1). A significant reduction in mortality was observed in the ATIII group (0 of 7) compared with the placebo group (4 of 6) (P<0.05, chi2 test) a significant reduction of pulmonary hypertension (placebo, 42.0+/-11. 1 mm Hg; ATIII, 23.6+/-7.5 mm Hg, P<0.05), but no effect on systemic hypotension, was noted in the ATIII group. It was thus concluded that modulation of the procoagulatory state by substitution of ATIII results in a late beneficial antiinflammatory effect in this model of septic shock.

Development of an anti-bleeding agent for recombinant hirudin induced skin bleeding in the pig.

Recombinant hirudin (rH) is a highly specific thrombin inhibitor which is under clinical investigation for various thrombotic disorders. However, one of its potential risks during clinical use might be hemorrhage, especially when combined with other agents interfering with the coagulation system like antiplatelet or fibrinolytic agents. In this experimental study we investigated whether Haemate, a von Willebrand Factor (vWF) and factor VIII containing product, could correct rH/aspirin induced bleeding in an experimental pig study. Skin bleeding time was evaluated in three open, placebo-controlled, randomized studies following comparable designs. A total of 62 animals were given a short-term infusion of aspirin (20 mg/kg) followed by a three-hour infusion of a high or low dose (0.3 or 0.5 mg/kg/h) of rH. At cessation of rH infusion, animals were allocated to treatment with either Haemate (30 FVIII U/kg) or the recombinant factor VIII, Helixate, which is devoid of vWF. The skin bleeding time (SBT, given as times of baseline) as measured four hours after the start of the rH infusion was defined as the prospective endpoint. In study 1 (low dose rH + Haemate) 4 h SBT was 2.18 (placebo) and 1.61 (Haemate, p = 0.0111). In study No. 2 (high dose rH + Haemate) SBT was 2.58 in placebo and 1.73 in Haemate (p = 0.0001). No significant difference between placebo and treatment were detected in study No. 3 (low dose rH + Helixate). Haemate but not Helixate significantly decreased bleeding time as compared to placebo at termination of the study (7 hours) which was defined as the secondary endpoint. No effect on either aPTT nor rH plasma levels were observed with any of the study drugs. It was concluded that Haemate decreases excess bleeding induced by rH/aspirin treatment without altering rH's anticoagulant effect.

Antithrombin III in animal models of sepsis and organ failure.

Antithrombin III (AT III) is the physiological inhibitor of thrombin and other serine proteases of the clotting cascade. In the development of sepsis, septic shock and organ failure, the plasma levels of AT III decrease considerably, suggesting the concept of a substitution therapy with the inhibitor. A decrease of AT III plasma levels might also be associated with other pathological disorders like trauma, burns, pancreatitis or preclampsia. Activation of coagulation and consumption of AT III is the consequence of a generalized inflammation called SIRS (systemic inflammatory response syndrome). The clotting cascade is also frequently activated after organ transplantation, especially if organs are grafted between different species (xenotransplantation). During the past years AT III has been investigated in numerous corresponding disease models in different animal species which will be reviewed here. The bulk of evidence suggests, that AT III substitution reduces morbidity and mortality in the diseased animals. While gaining more experience with AT III, the concept of substitution therapy to maximal baseline plasma levels (100%) appears to become insufficient. Evidence from clinical and preclinical studies now suggests to adjust the AT III plasma levels to about 200%, i.e., doubling the normal value. During the last few years several authors proposed that AT III might not only be an anti-thrombotic agent, but to have in addition an anti-inflammatory effect.

Combined treatment with C1 esterase inhibitor and antithrombin III improves survival in severe acute experimental pancreatitis.

BACKGROUND: Patients with severe acute pancreatitis die of complications closely related to the systemic activation of protease cascades. AIM: To examine the effects of human C1 esterase inhibitor (C1 INH) and antithrombin III (AT III) on two experimental models of acute pancreatitis. METHODS: Oedematous pancreatitis was induced by continuous intravenous infusion of caerulein and haemorrhagic pancreatitis by retrograde injection of sodium taurocholate into the biliopancreatic duct. C1 INH and AT III were given intravenously, either before or after the induction of pancreatitis. Treatment with C1 INH and AT III had no beneficial effect on oedematous pancreatitis. On the other hand, combined C1 INH and AT III therapy improved the survival in haemorrhagic pancreatitis compared with treatment with human serum albumin. This reduction in mortality was found regardless of whether the treatment was given prophylactically or therapeutically. CONCLUSIONS: Treatment with C1 INH and AT III represents a promising therapeutic concept for patients with severe haemorrhagic pancreatitis.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide.

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na(+)-dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na(+)-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.

First-pass elimination of a peptidomimetic thrombin inhibitor is due to carrier-mediated uptake by the liver. Interaction with bile acid transport systems.

CRC 220 (4-methoxy-2, 3, 6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinophenylalanyl -piperidide) is a competitive peptide-based trombin inhibitor with high affinity to human alpha-thrombin (Ki 2.5 nM). The amphiphilic compound exhibits virtually no systemic bioavailability despite proteolytic stability and proven enteral absorption. After intravenous application (V. jejunalis) in rats CRC 220 is almost completely excreted into bile. Simultaneous administration of bile acids considerably decreases this first-pass elimination. CRC 220 is extensively taken up in isolated rat hepatocytes by a saturable carrier-mediated transport with Km 23.7 microM and Vmax 775 pmol x mg-1 x min-1. A large part of this transport is energy-dependent. At temperatures above 20 degrees C, the uptake is accelerated exponentially. The activation energy amounts to 82 kj/mol. A minor portion of CRC 220 uptake occurs by physical diffusion with a permeability coefficient of 7.83 x 10(-7) cm/sec at 12 degrees C. Sodium ions energize CRC 220 uptake. Replacement of sodium by choline or lithium decreases the transport rate of 23-40%. In addition, a negative membrane potential facilitates the uptake. CRC 220 transport is only observed in hepatocytes: it is absent in BHK, FAO, HepG2, HPCT 1E3, and HPCT 1E3-TC cells. In the presence of 4-amidinophenylalanine derivatives, CRC 220 uptake is considerably decreased. Inhibition also occurs with bile acids and bromosulfophthalein, but less with bumetanide. Because CRC 220 inhibits bile acid uptake into hepatocytes and vice versa, the results suggest that the first-pass elimination of this amphiphilic thrombin inhibitor is due to an active carrier-mediated transport process in the basolateral plasma membrane of rat hepatocytes, and that this transport occurs via a bile acid transport system.

Influence of recombinant hirudin on tissue-factor-induced activation of coagulation in rabbits.

Uncontrolled activation of the tissue-factor (TF)-dependent extrinsic pathway of coagulation can lead to severe impairment of the hemostatic balance. AS thrombin plays the central role in the initiation of clotting, we used the highly specific thrombin inhibitor recombinant hirudin to prevent TF-induced coagulation activation in a rabbit model. Infusion of 0.5 micrograms.kg-1.h-1 TF in rabbits for 7 h led to a decrease in fibrinogen and platelets, to an increase in fibrin monomers and to a prolongation of TT, aPTT and PT. Recombinant hirudin was administered in doses of 0.5, 1 and 2 mg.kg-1 body weight (intravenous bolus), the protocol included a pre-TF (recombinant hirudin given at t = 0) and a post-TF study group (recombinant hirudin given at t = 2 h after the start of the TF infusion). Fibrinogen plasma levels, platelet counts and recombinant hirudin plasma levels were measured at baseline (t = 0) at 0.5, 1, 2, 3, 4, 5, 6, and 7 h; the deceleration rate of fibrinogen and platelets per hour was calculated for the control and the recombinant-hirudin-treated groups. The deceleration rate for fibrinogen in the TF group was -0.227 g.l-1.h-1 and was reduced by recombinant hirudin to -0.119, -0.116 and -0.095 g.1-1.h-1 for 0.5, 1 or 2 mg.kg-1, respectively (significant differences to control group, Jonckheere-Terpstra test). The inhibitor similarly prevented the decrease of platelets dose-dependently. Recombinant hirudin was cleared from plasma with a terminal half-life of about 100 min; however, even after its clearance from plasma, recombinant hirudin significantly prevented the fibrinogen and platelet drop. Recombinant hirudin was effective when given in the pre-TF as well as in the post-TF phase.

Reduction of r-hirudin induced bleeding in pigs by the administration of von Willebrand factor.

To prevent r-hirudin induced excess bleeding an animal model was established in pigs for the investigation of an anti-bleeding strategy. We used the Simplate® device to monitor skin bleeding time (SBT) at the inner site of the ear. r-Hirudin infused in a dose of 0.3 mag per h induced a prominent increase of SBT. The aim of our studies was to reverse r-hirudin induced bleeding by enhancing platelet adhesion to the endothelium via the administration of von Willebrand Factor (vWF). Pigs were treated with vWF containing solutions (Haemate® and a vWF-concentrate) at 3h after the start of the r-hirudin infusion. Both compounds suppressed SBT 1h after administration and significantly prevented bleeding until the termination of the experiment. SBT values (given in times of baseline) in the placebo group were 3.32 ± 0.9, 1.51 ± 0.14 in the Haemate® and 1.85 ± 0.42 in the vWF concentrate group (P = 0.008 or 0.032, in a two-sided Kruskall-Wallis-test). Coagulation parameters (aPTT, PT) were unaltered by the treatment, as were the r-hirudin plasma levels suggesting that vWF is not an antidote in its strict sense. It is concluded that vWF reverses bleeding without altering the anticoagulant effect of r-hirudin. Addition of 20 mg/kg per h aspirin resulted in a further increase of SBT. Aspirin, moreover, suppressed platelet aggregation but did not alter platelet counts. In a further study, bleeding induced by r-hirudin and aspirin was antagonized by Haemate® (66 Ukg i.v. bolus + 187 Ukg per h for 2 h infusion) and a significant reduction of bleeding time occurred.