Coagulation problems in liver disease.

Three topics are addressed in this manuscript: (1) the causes of abnormal hemostasis in cirrhosis; (2) the evaluation of hemostasis in cirrhotic patients before invasive procedures or in the presence of bleeding; and (3) the assessment of the effect of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) on hemostatic function in cirrhotic patients. Laboratory experiments are described that could enhance the understanding of the effect of rFVIIa on blood coagulation during hemostasis in cirrhotic patients.

A novel clotting assay for quantitation of plasma prothrombin (factor II) using Echis multisquamatus venom.

Measuring plasma prothrombin activity seems useful for evaluating thrombotic risk and managing oral anticoagulant therapy as an adjunct to the international normalized ratio. Therefore, we designed a new plasma prothrombin assay based on the ability of Echis multisquamatus venom to activate prothrombin with only calcium as a cofactor. In this assay, 1 part of undiluted citrated plasma is added to 5 parts of a venom reagent and the clotting time is measured. The assay's advantages are that dilution of the test plasma is required only when prothrombin activity exceeds 100%, a single standard curve can be used over months for a given batch of stock reagent, and barium-adsorbed plasma is used for dilution of test plasma and construction of the standard curve, thus eliminating the need for prothrombin-deficient plasma. However, one should be aware of the following: (1) test samples must contain at least 200 mg/dL fibrinogen; and (2) when prothrombin concentrations were below 50%, the venom-based assay often gave values up to 10% higher than the thromboplastin-based assay. Values obtained in 262 plasma samples tested with the venom-based assay and with a thromboplastin-based prothrombin assay correlated well (r2 = 0.93).

Hemostatic factors in rabbit limb lymph: relationship to mechanisms regulating extravascular coagulation.

Mechanisms regulating extravascular coagulation in interstitial fluids of peripheral tissues are poorly understood, since measurements of hemostatic factors in these fluids are unavailable. Because lymph from a body region reflects the composition of its interstitial fluid, we measured hemostatic factors in limb lymph of rabbits both as activity and as antigen. Mean lymph-to-plasma activity ratios were the following: fibrinogen, 0.28; prothrombin, 0.26; factor X, 0.27; factor VII, 0.17; and factors V and VIII, 0.08. All lymph fibrinogen was clottable; fibrin degradation products were absent. Lymph von Willebrand factor antigen was < 10% of plasma antigen and consisted primarily of lower molecular weight multimers. Mean lymph-to-plasma activity ratio for antithrombin was 0.38 and for tissue factor pathway inhibitor the ratio was 0.40. Low levels of antithrombin-factor Xa were measurable in lymph. The data are compatible with a basal factor VIIa-tissue factor-catalyzed extravascular activation of factor X that is prevented from progressing to generation of fibrin in limb interstitial fluid and lymph by low levels of factor VIII and factor V and by the inhibitory activity of antithrombin and tissue factor pathway inhibitor.

A Protac-based screening test for activated protein C-resistant factor Va and other defects of the protein C anticoagulant pathway.

We have standardized a simple screening test for abnormalities of the protein C anticoagulant system. The test is basically a modified prothrombin time in which one aliquot of a test plasma is incubated for 3 min at 37 degrees C with Protac and another is incubated with buffer. During the incubation the Protac activates both protein C and factor V. The plasmas are then clotted with thromboplastin plus Ca2+, and the clotting time difference reflects the ability of the activated protein C (APC) to inactivate factor Va. With the use of Thromboplastin C Plus as the activator, clotting time differences found in 31 normal subjects (10.4 +/- 3.5 s, mean +/- 2SD) were distinct from clotting time differences found in 57 of 58 subjects with established APC-resistant factor Va (3.6 +/- 3.0 s). In addition, the Protac-based test detected six of seven patients with isolated protein C deficiency and 20 of 28 patients with isolated protein S deficiency. Because of the reported high prevalence of heterozygous APC-resistant factor Va in Caucasian populations, it should be particularly useful in determining whether this genetic risk is present in individuals who have experienced or are at increased environmental risk of venous thrombosis.

Correction of excessive anticoagulation with low-dose oral vitamin K1.

BACKGROUND: Despite earlier acceptance of oral vitamin K1 (phytonadione) for the treatment of excessive anticoagulation, some recent guidelines do not recommend its use. OBJECTIVE: To reevaluate the efficacy of oral vitamin K1 in correcting excessive anticoagulation. DESIGN: Case series. SETTING: Anticoagulation clinics at two university medical centers. PATIENTS: 81 outpatients who had an international normalized ratio (INR) greater than 5.0 but did not have significant bleeding. INTERVENTIONS: Withholding 1 or 2 doses of warfarin, administering 2.5 mg of oral vitamin K1, measuring the INR after 24 to 48 hours, and adjusting the warfarin dose. MEASUREMENTS: INRs were obtained from a portable capillary fingerstick monitor or from an automated photooptical coagulometer. RESULTS: In 68 of 71 patients (96%), oral vitamin K1 lowered the INR from between 5.0 and 10.0 to less than 5.0 without inducing resistance to further anticoagulation. CONCLUSIONS: Withholding 1 or 2 doses of warfarin and administering 2.5 mg of oral vitamin K1 is a reliable, safe, and inexpensive way to rapidly correct excessive anticoagulation (INR > 5.0) in patients who do not have serious bleeding episodes and have an INR of less than 10.0.

Reliability of a modified tissue factor-dependent factor V assay for activated protein C resistant factor Va using a calcium-containing thromboplastin.

The original tissue factor-dependent factor V assay for activated protein C resistant factor Va (Blood 1995; 85: 1704-1711) has been modified to use a calcium containing thromboplastin and to express results as an observed to expected ratio (Obs/Exp.). The latter permits establishing a normal range independent of variations due to differences in reagents. Comparing Obs/Exp ratios with DNA analysis in 72 persons revealed that an Obs/Exp ratio of 0.6 distinguished without overlap normals from heterozygotes for FV R506Q. Three homozygotes had a ratio of < 0.1. Application of this Obs/Exp cut-off ratio of 0.6 to a total of 226 plasma samples tested to date discriminated without overlap between normals and heterozygotes. We conclude that this assay-readily adaptable to any dedicated coagulation laboratory and capable of yielding reliable results in all clinical circumstances in which testing is indicated-can distinguish between normals and heterozygotes for the FV R506Q mutation without the need for confirmatory DNA analysis.

The effect of immunodepletion of antithrombin III on the response of rabbits to Russell's viper venom-induced activation of factor X.

Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X-activating fraction of Russell's viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/ TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.

Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid.

We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

Plasma PIVKA proteins in rabbits given warfarin.

The presence of partially carboxylated forms of the vitamin K dependent coagulation factors (PIVKA) was evaluated in the plasma of rabbits treated with warfarin. Excess antigen over activity as measured in rabbit specific assays was taken as evidence for PIVKA. Our data confirm a previous report of the absence of plasma PIVKA prothrombin. In contrast, plasma PIVKA factors VII, IX, and X were demonstrable. A striking excess of plasma factor IX antigen over activity was measured and a large fraction of the factor IX antigen persisted in the plasma after its adsorption with barium citrate.

Studies of the activation of factor VII bound to tissue factor.

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I-factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

Cells and the activation of factor VII.

Binding of factor VII to tissue factor (TF) present on or released from cells initiates TF-dependent coagulation in vivo. Earlier data obtained with relipidated TF suspensions led to the hypothesis that all factor VII bound to limiting TF sites exposed after tissue injury could be rapidly activated to VIIa/TF complexes by low concentrations of factor Xa, IXa, and VIIa. However, newer data on the activation of factor VII bound to TF expressed on cultured cell monolayers support the hypothesis that not all VII/TF complexes formed at a site of tissue injury are readily activated to VIIa/TF complexes during hemostasis. This is possibly related to why recombinant factor VIIa can bypass impaired IXa/VIIIa/phospholipid complex function in hemostasis.

Use of a generally applicable tissue factor--dependent factor V assay to detect activated protein C-resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant.

The original activated partial thromboplastin time-based assay for activated protein C (APC)-resistant factor Va (FVa) requires carefully prepared fresh plasma and cannot be used in patients receiving warfarin or in patients with antiphospholipid antibodies. A new test is described here that circumvents these limitations and distinguishes without overlap heterozygotes for APC-resistant FVa from persons with normal FV. A diluted test plasma is incubated with an FV-deficient substrate plasma and tissue factor and then clotted with Ca2+ or Ca2+ plus APC. Test results are independent of the FV level or the dilution of the test plasma used. Of 39 controls, 37 gave normal results. Two controls (5%) gave results indicative of APC resistant FVa and on DNA analysis were found to be heterozygous for FV R506Q. Twenty of 21 randomly selected patients receiving warfarin gave normal results. In the single patient with abnormal results, heterozygous FV R506Q was confirmed by DNA analysis. Two of 15 patients with protein S deficiency and 5 of 29 patients with a lupus anticoagulant had abnormal results. APC resistance caused by FV R506Q was confirmed in the five of these seven patients available for DNA analysis. APC-resistant FVa was also detected in 10 of 21 (46%) stored plasma from unrelated patients with venous thrombosis and negative earlier evaluation for a lupus anticoagulant or a deficiency of protein C, protein S, or antithrombin, which confirms a high incidence of this defect among patients with venous thrombosis.

Differences in the interactions of lupus anticoagulant IgG with human prothrombin and bovine prothrombin.

Lupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin that the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell's viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing > 50% of inhibition. In contrast, only one LA IgG markedly inhibited (> 50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence suggestive of activation of the intrinsic pathway of blood coagulation after injection of factor Xa/phospholipid into rabbits.

The present study was carried out to extend an earlier observation from this laboratory that mean plasma factor X levels fell by about 15% after the injection into rabbits of a formed factor Xa/phospholipid complex that caused only minimal intravascular coagulation. We have now injected rabbits with formulations of factor Xa/phospholipid that caused considerable intravascular coagulation, as documented by substantial falls in fibrinogen, factor V, and factor VIII and a fall in plasma prothrombin activity of about 15% to 20% of the initial level. Mean plasma factor X activity fell by about 30% of the initial level. Factors participating in the intrinsic coagulation pathway--XII, XI, and IX--all fell by about 50% after injection of a complex made with 16.3 pmol factor Xa and 80 nmol phospholipid per 1 kg body wt and by about 35% after injection of a complex made with 32.6 pmol factor Xa and 40 nmol phospholipid per 1 kg body wt. In contrast, total plasma factor VII activity did not change, and specific plasma factor VIIa levels, which were lower than those measured in human plasma, did not rise after injection of factor Xa/phospholipid. The data are compatible with the hypothesis that factor Xa/phospholipid-induced generation of thrombin in vivo leads to factor XII-dependent activation of the intrinsic pathway of coagulation that results in significant activation of factor X. Further testing of this hypothesis appears warranted.

Evidence for an essential role of tissue factor dependent blood coagulation in the pathogenesis of the local Shwartzman reaction.

Adherence and aggregation of leukocytes within the vessels of a prepared skin site has been shown to be essential for the pathogenesis of the local Shwartzman reaction (LSR) (Argenbright and Barton: J Clin Invest 89: 259, 1992). We have now performed experiments in rabbits to evaluate whether coagulation within the vessels of a prepared site is a second requirement for the reaction. Skin sites were prepared by an intradermal injection of endotoxin 24 hours before a provoking intravenous injection of endotoxin. Thirteen control rabbits all developed the LSR. Seven of 12 rabbits given warfarin to achieve anticoagulation approximating that used therapeutically in humans before the provoking injection were protected against the LSR (p = 0.003). Five of nine rabbits given anti-rabbit factor X IgG before the provoking injection to yield mean values in individual rabbits of between 7% and 18% plasma factor X activity were protected against the LSR (p = 0.009). Six of 11 rabbits given anti-rabbit factor VII IgG before the provoking injection to yield mean values in individual rabbits of between < 0.5% and 2.2% were protected against the LSR (p = 0.007). Four rabbits failed to develop the LSR at an endotoxin-prepared skin site when an infusion of tissue factor (TF) causing substantial intravascular coagulation was substituted for a provoking injection of endotoxin. It would appear that two events are required for the pathogenesis of the LSR provoked by endotoxin: formation of aggregated masses of WBC in the prepared skin vessels and deposition of fibrin due to TF-initiated coagulation.