Balance between von Willebrand factor and ADAMTS13 following major partial hepatectomy.

BACKGROUND: Conventional coagulation tests are frequently prolonged after liver surgery, suggesting a postoperative bleeding tendency. At the same time, thrombotic complications following partial hepatectomy (PH) are not uncommon. Little is known about changes in the platelet adhesive protein von Willebrand factor (VWF) and its cleaving protease a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) following a PH. METHODS: Plasma samples were collected before and after PH and pylorus-preserving pancreaticoduodenectomy (PPPD), and from 24 healthy individuals. Plasma levels of VWF and ADAMTS13, VWF activity and VWF-dependent platelet adhesion were measured, and compared between the groups. RESULTS: Median (i.q.r.) VWF levels increased more after PH (17 patients) than following PPPD (10), reaching the highest level on postoperative day (POD) 3 (570 (473-656) versus 354 (305-476) per cent respectively; P = 0·009). VWF levels remained raised on POD 30. A decrease in median (i.q.r.) ADAMTS13 activity was observed for both patient groups, reaching the lowest level on POD 7 (24 (16-32) versus 38 (23-66) per cent for PH and PPPD respectively; P = 0·049), and levels remained significantly reduced at POD 30. VWF activity was significantly higher on day 7 following PH compared with PPPD (median (i.q.r.) 517 (440-742) versus 385 (322-484) per cent respectively; P = 0·009), and remained increased at POD 30. VWF-dependent platelet adhesion under conditions of flow was increased until POD 30 in patients after PH and PPPD, but was more pronounced in the PH group. CONCLUSION: There are changes in the balance between VWF and ADAMTS13 levels and activity in patients after both PH and PPPD. Changes in the VWF-ADAMTS13 axis were more pronounced and of longer duration after PH than following PPPD.

Procoagulant changes in fibrin clot structure in patients with cirrhosis are associated with oxidative modifications of fibrinogen.

UNLABELLED: Essentials Patients with cirrhosis have hemostatic changes, which may contribute to a risk of thrombosis. This in vitro study compares clot formation and structure between patients and healthy subjects. Clot formation is delayed in patients; ultimately, however, clot permeability is decreased. The thrombogenic structure of fibrin clots may contribute to the thrombotic risk in cirrhosis. ABSTRACT: Background and Objectives Patients with cirrhosis can be at risk of thrombotic complications due to an imbalance between hemostatic components. However, little is known on how the disease affects clot generation or how alterations in the structure of fibrin clots may affect the hemostatic function of these patients. Methods We investigated the formation and structure of clots generated with plasma and purified fibrinogen of 42 patients with cirrhosis. Clots generated with plasma and fibrinogen of 29 healthy volunteers were studied for comparison. Clot formation and structure were assessed by turbidity, permeation studies, confocal laser and scanning electron microscopy (SEM). The extent of fibrinogen oxidation was assessed by measuring the carbonyl content of purified fibrinogen samples. Results Tissue factor and thrombin-induced clotting of plasma was delayed in patients. The clotting rate was also decreased, but change in turbidity, fibrin density and fiber thickness were largely comparable to healthy volunteers. Conversely, clot permeability was significantly decreased in patients. When clots were generated with purified fibrinogen, differences in clot formation and structure similar to those in plasma were found. The carbonyl content was increased in patient fibrinogen and correlated with disease severity and clot permeability. Conclusions Delayed clot formation in cirrhosis ultimately results in decreased clot permeability. Similar alterations in clots generated with purified fibrinogen suggest that modifications of the molecule are (partly) responsible. Taken together, these findings are indicative of hypercoagulable features of clots of patients with cirrhosis, which may explain the increased risk of thrombosis associated with this condition.

Ex vivo addition of fibrinogen concentrate improves the fibrin network structure in plasma samples taken during liver transplantation.

BACKGROUND: Optimal hemostatic management during orthotopic liver transplantation (OLT) remains a challenge. The cause of bleeding during OLT is multifactorial, and may include hemostatic imbalance. Fibrinogen concentrates are increasingly being used to control perioperative bleeding during OLT. However, administration is based on arbitrary thresholds of fibrinogen levels. Importantly, studies on fibrin clot structure during OLT are lacking. OBJECTIVE: We determined the hemostatic efficacy of fibrinogen concentrate in correcting the fibrin structure. METHODS: Plasma samples taken at various times during OLT from 15 patients and 15 healthy controls were spiked with 1 g L(-1) fibrinogen concentrate or saline. Turbidity, fibrin fiber density and permeability of the fibrin clots were assessed. RESULTS: Clotting rate and turbidity were significantly decreased at the start of surgery, and decreased even further during surgery. Addition of fibrinogen significantly increased the clotting rate and turbidity at all time points, but did not normalize it. Fibrin density was significantly reduced after reperfusion as compared with the density at the start of surgery and in healthy controls. Fibrin density improved significantly after addition of fibrinogen in samples taken at the start of surgery and after reperfusion. The severely impaired polymerization and decreased density after reperfusion were accompanied by significantly increased permeability of the clot as compared with the start of surgery and in controls, which was completely restored after addition of fibrinogen. CONCLUSIONS: Ex vivo addition of fibrinogen concentrate during OLT substantially improves the structural properties of the fibrin clot, which, particularly after reperfusion, shows hypocoagulable features. These data support the use of fibrinogen concentrate to control bleeding complications during OLT.

Development of a Hyperactive Primary Hemostatic System During Off-Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13.

An unbalance between the platelet-adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 is a risk factor for thrombosis. Here, we assessed levels and functionality of VWF and ADAMTS13 in patients undergoing off-pump lung transplantation. We analyzed plasma of 10 patients and distinguished lung transplantation-specific effects from those generally accompanying open-chest surgeries by comparing results with 11 patients undergoing off-pump coronary bypass graft (CABG) surgery. Forty healthy volunteers were included for reference values. VWF antigen levels as well as the VWF ristocetin cofactor activity/VWF antigen ratio increased during lung transplantation and after CABG surgery. An increase in VWF propeptide levels was paralleled by a decrease in ADAMTS13 activity. This was more pronounced during lung transplantation. Similarly, the capacity of plasma to support platelet aggregation under shear flow conditions in vitro was more increased during lung transplantation. The proportion of high molecular weight VWF multimers was elevated in both groups without evidence for ultra-large VWF. VWF's collagen binding activity remained unchanged. In conclusion, a hyperactive primary hemostatic system develops during lung transplantation resulting both from a pronounced (functional) increase of the VWF molecule and decrease of ADAMTS13. This may increase the risk of platelet thrombosis within the allograft.

Hypercoagulability following major partial liver resection - detected by thrombomodulin-modified thrombin generation testing.

BACKGROUND: Conventional coagulation tests are frequently prolonged after liver surgery, suggesting a post-operative hypocoagulability. However, these tests are unreliable for assessment of the haemostatic status in these patients. In contrast, thrombin generation testing measures the true balance between pro- and anti-coagulant factors. AIM: To study the perioperative coagulation status in patients undergoing hemi-hepatectomy using thrombin generation assays. METHODS: We examined thrombin generation profiles in serial plasma samples taken from seventeen patients undergoing right hemi-hepatectomy. Results were compared to ten patients undergoing pancreatic resection and twenty-four healthy volunteers. In addition, we measured conventional coagulation tests and plasma levels of several haemostatic proteins. RESULTS: Following liver resection, the endogenous thrombin potential (ETP) slightly decreased until post-operative day 7. However, in the presence of thrombomodulin, the ETP increased [from 542 nM*min (417-694) at baseline to 845 nM*min (789-1050) on post-operative day 3] to values higher than that in healthy subjects (558 nM*min (390-680); P < 0.001), which contrasts with substantially prolonged PT levels. Normal to decreased thrombin generation was observed following pancreatic resection. Thrombin generation was only slightly affected by thrombomodulin after hemi-hepatectomy, while thrombin generation in healthy subjects decreased profoundly upon addition of thrombomodulin. This hypercoagulability following liver resection may be explained by decreased levels of protein C, S, and antithrombin and by elevated levels of factor VIII. CONCLUSIONS: Thrombin generation in the presence of thrombomodulin revealed hypercoagulability in patients following liver resection. These results support the recently advocated restrictive use of plasma during liver resection and the exploration of more extensive use of post-operative thrombosis prophylaxis.

Sustained pro-haemostatic activity of rFVIIa in plasma and platelets in non-bleeding pigs may explain the efficacy of a once-daily prophylaxis in humans.

Recombinant factor VIIa (rFVIIa) is registered for treatment of inhibitor-complicated haemophilia, and a once-daily prophylactic administration of rFVIIa is successful in reducing the number of bleeding events. This suggests that a single rFVIIa dose has a pro-haemostatic effect up to 24 hours (h), which is difficult to explain given its half-life of 2 h. In this study, six pigs received a 90 µg/kg rFVIIa bolus. Plasma was collected and platelets were isolated at various time points up to 48 h, and analysed for FVIIa levels and associated haemostatic activity. Elevated plasma FVIIa levels were detected up to 24 h post-administration (36 (32-56) mU/ml [median (interquartile range [IQR]), 24 h] vs 2 (2-14) mU/ml [baseline]). Corresponding prothrombin time (PT) values remained shortened compared to baseline until 24 h post-administration (9.4 (9.3-9.9) seconds (s) [24 h] vs 10.5 (10.2-11.0) s [baseline], p ≤0.01). The lag time in thrombin generation testing as well as clotting times in plasma-based assays were shortened up to 12 or 24 h post-administration, respectively (lag times 1.8 (1.7-2.1) minutes (min) [12 h] vs 2.3 (2.3-2.6) min [baseline], p ≤0.01 and clotting times 3.8 (3.2-3.9) min [24 h] vs 5.2 (4.6-5.5) min [baseline], p ≤0.001). Platelet FVIIa levels were elevated up to 48 h (7.7 (3.4-9.0) ng VIIa/mg actin [48 h] vs 2.5 (0.7-4.8) ng VIIa/mg actin [baseline]). In conclusion, elevated and haemostatically active plasma and platelet FVIIa levels are detectable up to 24-48 h following rFVIIa administration in pigs. This prolonged pro-haemostatic effect of FVIIa may explain the prophylactic efficacy of a once-daily rFVIIa treatment.

In vitro effects of proteases in human pancreatic juice on stability of liquid and carrier-bound fibrin sealants.

BACKGROUND: Fibrin sealants are used in pancreatic surgery to prevent leakage of pancreatic fluid and reduce associated complications. The efficacy of this approach is unclear. METHODS: Fibrin clots were generated in vitro from two commercially available liquid fibrin sealants (Tissucol Duo® and Evicel®) and the carrier-bound fibrin sealant Tachosil®, and exposed to normal saline or human pancreatic fluid. Stability of the sealants was assessed by release of the fibrin and collagen degradation products, D-dimer and hydroxyproline. The effect of protease inhibitors on sealant breakdown was assessed. RESULTS: Clots generated from liquid fibrin sealants degraded rapidly in pancreatic fluid, but not in normal saline. D-dimer release from fibrin clots by pancreatic fluid was approximately 1700 µg/ml after 24 h and less than 20 µg/ml by saline. Pancreatic fluid, but not normal saline, degraded both the fibrin and collagen component of Tachosil®. After 6 h, mean(s.e.m.) D-dimer levels in pancreatic fluid exposed to Tachosil® were 850(183) ng/ml, compared with 60(6) ng/ml in normal saline. The mean(s.e.m.) hydroxyproline concentration in pancreatic fluid was 497(17) µg/ml after a 24-h exposure to Tachosil®, compared with 26(12) µg/ml in normal saline. Protease inhibitors significantly inhibited breakdown of liquid sealants (D-dimer levels less than 50 µg/ml after 24 h) and Tachosil® (D-dimer release 179(12) ng/ml at 6 h; hydroxyproline release 181(29) µg/ml at 24 h). CONCLUSION: Proteases in pancreatic juice effectively degrade both liquid and carrier-bound fibrin sealants in vitro. The use of these products in pancreatic surgery with the aim of preventing leakage of pancreatic fluid is not supported by this experimental study.

TAFI deficiency promotes liver damage in murine models of liver failure through defective down-regulation of hepatic inflammation.

Emerging evidence indicates that various haemostatic components can regulate the progression of liver disease. Thrombin-activatable fibrinolysis inhibitor (TAFI) possesses anti-inflammatory properties besides its anti-fibrinolytic function. Here, we investigated the contribution of TAFI to the progression of disease in murine models of chronic and acute liver failure. Chronic carbon tetrachloride (CCL4) administration induced liver damage and fibrosis both in TAFI knockout (TAFI-/-) mice and wild-type controls. Smooth muscle actin-α (α-SMA) content of liver tissue was significantly increased after 1 and 3 weeks, and pro-collagen α1 expression was significantly increased after 3 and 6 weeks in TAFI-/- mice. TAFI-/- mice showed significantly elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after 3 weeks of CCL4. Neutrophil influx was significantly increased in TAFI-/- mice after 6 weeks of CCL4. No difference in hepatic fibrin deposition between TAFI-/- and wild-types was observed. After acetaminophen intoxication, necrosis was significantly increased in TAFI-/- mice at 24 hours (h) after injection. AST and ALT levels were decreased at 2 and 6 h after acetaminophen injection in TAFI-/- mice, but were significantly higher in the TAFI-/- mice at 24 h. Similarly, hepatic fibrin deposition was decreased at 6 h in TAFI-/- mice, but was comparable to wild-types at 24 h after injection. In conclusion, TAFI deficiency results in accelerated fibrogenesis and increased liver damage in murine models of chronic and acute liver disease, which may be related to increased inflammation.

Intact thrombin generation and decreased fibrinolytic capacity in patients with acute liver injury or acute liver failure.

BACKGROUND: It has been well established that hemostatic potential in patients with chronic liver disease is in a rebalanced status due to a concomitant decrease in pro- and antihemostatic drivers. The hemostatic changes in patients with acute liver injury/failure (ALI/ALF) are similar but not identical to the changes in patients with chronic liver disease and have not been studied in great detail. OBJECTIVE: To assess thrombin generation and fibrinolytic potential in patients with ALI/ALF. METHODS: We performed thrombin generation tests and clot lysis assays in platelet-poor plasma from 50 patients with ALI/ALF. Results were compared with values obtained in plasma from 40 healthy volunteers. RESULTS AND CONCLUSION: The thrombin generation capacity of plasma from patients with ALI/ALF sampled on the day of admission to hospital was indistinguishable from that of healthy controls, provided thrombomodulin was added to the test mixture. Fibrinolytic capacity was profoundly impaired in patients with ALI/ALF on admission (no lysis in 73.5% of patients, compared with 2.5% of the healthy controls), which was associated with decreased levels of the plasminogen and increased levels of plasminogen activator inhibitor type 1. The intact thrombin generating capacity and the hypofibrinolytic status persisted during the first week of admission. Patients with ALI/ALF have a normal thrombin generating capacity and a decreased capacity to remove fibrin clots. These results contrast with routine laboratory tests such as the PT/INR, which are by definition prolonged in patients with ALI/ALF and suggest a bleeding tendency.

Development of a severe von Willebrand factor/ADAMTS13 dysbalance during orthotopic liver transplantation.

Patients with liver disease show profound changes in their hemostatic system, which may further change during liver transplantation. We previously demonstrated that highly elevated levels of the platelet adhesive protein von Willebrand factor (VWF) in patients with cirrhosis lead to an increased VWF-dependent platelet deposition under flow as compared to healthy controls. In this study we examined VWF parameters during the course of liver transplantation. We collected serial plasma samples from 20 patients undergoing liver transplantation in which we determined plasma levels of VWF and the VWF-cleaving protease ADAMTS13. Furthermore, we performed functional tests of VWF-dependent platelet adhesion. We found persistently elevated levels of VWF during and after liver transplantation. The capacity of VWF to interact with platelets normalized during the course of transplantation, and flow-mediated VWF-dependent platelet adhesion remained at levels far exceeding those observed in healthy individuals during and after transplantation. Plasma levels of ADAMTS13 dropped during transplantation, and in four patients levels below 10% of normal were observed after reperfusion. We observed the development of a hyperreactive primary hemostatic system, as evidenced by high levels of fully functional VWF and a temporary ADAMTS13 deficiency, during liver transplantation, and speculate that these changes contribute to postoperative thrombotic complications.

Platelet adhesion to dimeric beta-glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibalpha and apolipoprotein E receptor 2'.

BACKGROUND: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (beta2GPI). Dimerized beta2GPI binds to apolipoprotein E receptor 2' (apoER2') on platelets and increases platelet adhesion to collagen under conditions of flow. AIM: To investigate whether the interaction between dimerized beta2GPI and platelets is sufficiently strong to resist shear stresses. METHODS: We studied the interaction of platelets with immobilized dimerized beta2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. RESULTS: We found that dimerized beta2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized beta2GPI was completely inhibited by the addition of soluble forms of both apoER2' and GPIbalpha, and the addition of receptor-associated protein and the removal of GPIbalpha from the platelet surface. GPIbalpha co-precipitated with apoER2', suggesting the presence of complexes between GPIbalpha and apoER2' on platelet membranes. The interaction between GPIbalpha and dimeric beta2GPI was of intermediate affinity (Kd = 180 nM) and Zn2+, but not Ca2+-dependent. Deletion of domain V from dimeric beta2GPI strongly reduced its binding to both GPIbalpha and apoER2'. Antibodies that inhibit the binding of thrombin to GPIbalpha inhibited platelet adhesion to dimeric beta2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbalpha had no effect. Dimeric beta2GPI showed reduced binding to low-sulfated GPIbalpha compared to the fully sulfated form. CONCLUSION: We show that platelets adhere to dimeric beta2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbalpha and apoER2'. These receptors are present in a complex on the platelet surface.

Recombinant factor VIIa enhances platelet adhesion and activation under flow conditions at normal and reduced platelet count.

BACKGROUND: Recombinant factor VIIa (rFVIIa), which was developed for treatment of inhibitor-complicated hemophilia, appears suitable as prohemostatic agent in other clinical disorders including patients with thrombocytopenia. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. OBJECTIVES: To determine the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet counts. METHODS: Washed platelets and red cells were combined to obtain plasma-free blood with different platelet counts. The reconstituted blood was perfused over a collagen- or fibrinogen-coated surface in the absence or presence of a thrombin generating system consisting of purified coagulation factors rFVIIa, factor (F)X and prothrombin. RESULTS: Addition of coagulation factors rFVIIa, FX and prothrombin to washed platelets and red cells enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10 000 microL(-1). rFVIIa-mediated thrombin generation enhanced the activation state of platelets as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. CONCLUSIONS: Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (i) enhancement of primary hemostasis and (ii) enhancement of procoagulant surface leading to elevated fibrin formation.

Recombinant factor VIIa reverses the in vitro and ex vivo anticoagulant and profibrinolytic effects of fondaparinux.

BACKGROUND: Fondaparinux is a synthetic pentasaccharide, which selectively inhibits coagulation factor (F) Xa, and is registered for prevention of venous thromboembolism following hip fracture, hip replacement, and knee replacement surgery. Recently, it was shown that recombinant FVIIa (rFVIIa) reverses anticoagulant effects of fondaparinux in healthy volunteers. OBJECTIVES: In this study, we have explored the in vitro and ex vivo effects of rFVIIa on clot formation and thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated down-regulation of fibrinolysis after fondaparinux administration. METHODS: In vitro clot lysis assays were performed in pooled normal plasma from healthy volunteers to which fondaparinux was added, and in serial samples from healthy volunteers who received a single bolus dose of fondaparinux, a single bolus dose of rFVIIa, or both. RESULTS AND CONCLUSIONS: Fondaparinux significantly delayed clot formation, and clot lysis was significantly increased due to decreased activation of TAFI. Addition of recombinant FVIIa corrected the inhibited clot formation induced by fondaparinux, and the acceleration of clot lysis was partially reversed. In vivo administration of fondaparinux (10 mg) to healthy volunteers similarly resulted in accelerated plasma clot lysis. Subsequent administration of rFVIIa (90 microg kg(-1)) normalized the clot lysis time up to 6 h postadministration. rFVIIa might be a good therapeutic option in patients treated with fondaparinux who develop bleeding complications, since both clot formation as well as fibrinolytic resistance are improved.