Rate Of Prothrombin Activation Research Articles

A Plasma-Based Assay to Measure the Susceptibility of Factor V(a) to Inhibition By TFPIα

Background: Coagulation factor V (FV) is the precursor of activated FV (FVa), which assembles with factor Xa (FXa) on phospholipid surfaces to form the prothrombinase complex, accelerating prothrombin activation >1000-fold. FV is activated by FXa or thrombin via limited proteolysis at Arg709, Arg1018 and Arg1545. These cleavages progressively expose the FXa-binding site, generating activation intermediates with increasing affinities for FXa. Recently, it has been shown that tissue factor pathway inhibitor α (TFPIα) inhibits both the activation of FV (by interfering with cleavage at Arg1545) and the ability of partially activated FV(a) species to enhance prothrombin activation. These effects are mediated by interactions of the C-terminus of TFPIα with an acidic region in the B-domain of FV as well as with the FV(a) heavy chain. The pathophysiological relevance of these novel anticoagulant activities of TFPIα is still unexplored, but evidence has been provided that prothrombinase complexes assembled with FV(a) Leiden are less susceptible to TFPIα inhibition. Moreover, FV splicing variants (FV-short) with increased affinity for TFPIα have recently been discovered in two unrelated families (from East Texas and Amsterdam, respectively) with bleeding tendencies.Rationale and Aim: The FV present in plasma from different individuals may differ in its sensitivity to inhibition by TFPIα, with potential implications for the risk of venous thrombosis or bleeding. Therefore, the aim of this study was to develop a plasma-based assay that measures the susceptibility of FV(a) to TFPIα inhibition.Methods: FV in 1/1000 diluted plasma was activated for 3 minutes with a suboptimal FXa concentration on 20/60/20 DOPS/DOPC/DOPE lipids in the presence or absence of a peptide mimicking the C-terminus of TFPIα (TFPIα C-term). Purified prothrombin and a chromogenic substrate for thrombin were then added, and the activity of the prothrombinase complex was monitored continuously up to 30 minutes. The parabolic absorbance curves were fitted to second-order polynomial equations and the rate of prothrombin activation was calculated from the coefficient of the x2-term. The assay outcome was expressed as residual prothrombinase ratio (RP-ratio), defined as the ratio between the rates of prothrombin activation obtained in the presence and absence of TFPIα C-term. The assay was validated using plasma from 4 FV Leiden homozygotes and 4 normal controls. In addition, we tested plasma from 3 members of the FV Amsterdam family (2 carriers of the mutation up-regulating FV-short Amsterdam and 1 non-carrier).Results: The rate of prothrombin activation in the absence of peptide was a function of plasma FV level and pre-incubation time, and was inhibited by TFPIα C-term in a dose-dependent manner. A pre-incubation time of 3 minutes and a peptide concentration of 100 nM, yielding an RP-ratio of 0.30 in normal pooled plasma, were chosen. The RP-ratio was independent of the plasma FV level in the 75-150% range. Moreover, control experiments indicated that, at this high dilution, the plasma background did not influence the assay outcome. The intra- and inter-assay coefficients of variation of the RP-ratio were 5.4% and 12%, respectively. FV Leiden homozygotes had higher RP-ratios than normal controls (0.45 ± 0.04 vs. 0.30 ± 0.03, p=0.002), indicating resistance to inhibition by TFPIα C-term. Differently, the 2 carriers of the FV Asterdam mutation, who express high levels of FV-short Amsterdam, had markedly reduced RP-ratios (0.18 and 0.16 vs. 0.29 in the non-carrier), as expected from the high affinity of FV-short Amsterdam for TFPIα.Conclusions: We have developed and validated an assay that measures the susceptibility of plasma FV(a) to inhibition by TFPIα. This assay can be used to test whether TFPIα-mediated inhibition of FV activation and prothrombinase activity differs for (genetically) different FV variants and whether it correlates with the risk of thrombosis or bleeding.Supported by grant 2014-1 from the Dutch Thrombosis Foundation. DisclosuresNo relevant conflicts of interest to declare.

Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG.Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography.Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket.We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma.Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresBos:Bayer Hemophilia Awards: Research Funding.

Effect of Zymogen Domains and Active Site Occupation on Activation of Prothrombin by von Willebrand Factor-binding Protein

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

ACTIVATION OF FACTOR V BY VENOM PROTEASES

Blood coagulation factor V is the nonenzymatic protein cofactor essential to the formation of thrombin. Activated factor V, FVa, is a cofactor of FXa in the so-called prothrombinase complex. Inclusion of FVa in this complex results in a more than 1,000-fold enhancement of the rate of prothrombin activation. Several animal venoms contain activities that have the capacity to activate human factor V. Besides activators of factor V, inactivating enzymes are also found in animal venoms. Of those venom proteases that affect FV activity, snake venoms of different families, including those of the Viperidae, Crotalidae, and Elapidae, have been most widely studied. Venom factor V activators have proven to be excellent tools in studying the structure–function relationship of factor V(a). In particular, one of the proteases of the Viperidae family, the factor V activator from the venom of Russell's viper (RVV-V) has been very helpful, because it is also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway.In this paper, we present an up-to-date overview of those venom proteases that possess the capacity to activate or inactivate coagulation factor V and, where possible, summarize structural and functional properties of these proteases. Furthermore, we provide a novel detailed three-dimensional homology model for RVV-V, the factor V activator from Russell's viper venom. This model will be helpful to explain the remarkable substrate specificity of RVV-V on the basis of its three-dimensional structure.

Aspirin and thrombinogenesis

Aspirin (ASA) not only inhibits platelet function, but increase in the rate of factor Va inactivation [12]. In patients also exerts additional anticoagulant actions. These actions are coupled with prevention of platelet activation by thromboxane (TX) A2, because only activated platelets are the source of prothrombinase and tenase complexes on their surface. Thus, ASA decreases thrombin formation in vitro and in vivo, in blood emerging from small skin incisions [1,2]. Yet, some patients experience recurrent arterial thrombotic events despite long-term preventive ASA therapy. Various reasons may lead to such episodes, one of them being so-called ASA resistance [3]. This phenomenon is still poorly defined; it could include the clinical inability of ASA to protect patients from cardiovascular events, as well as the failure of ASA to inhibit platelet function in a laboratory test system (e.g. platelet aggregation) [4], or to suppress overall TXA2 production [5]. There are at least two possible explanations for the ASA resistance phenomenon. One is hypercholesterolemia, probably by its virtue to change the physico-chemical properties of the platelet cell membrane [6]. Thus, cholesterol and atherogenic lipoproteins may increase platelet aggregability and TXA2 production [7,8], and support assembly of the prothrombinase complex and thrombin generation [9]. In patients with markedly elevated and borderline high cholesterol levels, ASA in a commonly used doses (75–300 mg/day) has negligible effect on thrombin formation, as measured by microquantitative analytical technique, at the site of microvascular injury [10,11]. On the other hand, 3 months treatment of such patients with simvastatin significantly depresses blood clotting in vivo, mainly through a reduction in thrombin generation [10,11]. Here, statin treatment seems to interfere with several interconnected steps leading to thrombin formation. They include the reduction in the rates of prothrombin activation, factor Va generation, fibrinogen cleavage, factor XIII activation, and a parallel

Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

Factor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5, 1.0 or 5 nM alpha-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM alpha-thrombin for 60 s) before adding 0.5, 1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Bovine Prothrombin as Monitored by the Clotting Assay

The activation of bovine prothrombin was studied with highly purified clotting factors and using a coagulation assay developed to look at the initial rate of prothrombin conversion as well as the conversion rate over a time course of 75 min. Activation of prothrombin by factor Xa alone was slow. The rate of prothrombin conversion increased markedly with the addition of each of the accessory components Ca2+, phospholipid and bovine factor V, respectively. With the complete prothrombinase complex comprising factor Xa, Ca2+, phospholipid and factor V, the rate increase was about 22,000-fold higher compared to the action of factor Xa and Ca2+ on prothrombin alone. The rates of thrombin formation obtained with activated factor X1 were only about 70% the values obtained with factor X2. The rate of prothrombin activation and the difference between the activities of the activated factors X1 and X2 are discussed.

Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators

Purified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombin-converting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Prothrombin clamart : prothrombin variant with defective arg 320-IIe cleavage by factor xa

An abnormal prothrombin has been detected in a 23 yr-old healthy female and her mother. Both patients appeared to be heterozygous for the abnormality, plasma prothrombin being 50 % of normal using the usual one stage assay, but normal when measured either by using Echis carinatus venom or by immunoassay. No abnormality in the immunoelectrophoretic pattern was observed. Prothrombin isolation on DEAE Sephadex failed to separate the abnormal population (prothrombin Clamart) from the normal one. The rates of prothrombin activation by factor Xa, in the presence or absence of phospholipids and/ or factor Va, were determined by measuring the production of both clotting and amidolytic activities. The thrombin generation rate from prothrombin isolated from the propositus plasma was 50 % slower than normal whatever the method of measurement and the composition of the activation mixture. Analysis of the final activation products by SDS polyacrylamide gel electrophoresis revealed that equal amounts of prethrombin 2 and thrombin had been formed. Prethrombin 2 Clamart was shown to be resistant to proteolysis upon further incubation with factor Xa, whereas it was readily converted to thrombin by Echis carinatus venom. Prothrombin Clamart appears to be characterized by an impairment of Arg 320-IIe cleavage by factor Xa.

Activation of prothrombin as measured by the conversion of Nalpha-benzoylarginine ethyl ester.

The activation of prothrombin has been studied by using highly purified preparations of activated factor X1 and activated factor X2, factor V and prothrombin. The rate of prothrombin activation was followed using an esterase assay involving the conversion of N alpha-benzoylarginine ethyl ester (BAEE) by thrombin generated in the course of prothrombin activation. The rate of thrombin generation increased by about 26000-fold when factor V and phospholipid were added to prothrombin, factor Xa and calcium. A comparison of the rates of thrombin formation obtained with activated factor X1 and activated factor X2 showed that activated factor X1 had only 70% of the biological activity of activated factor X2. Attempts to explain the rate of prothrombin activation and the difference between the activity of activated factor X1 and activated factor X2 are discussed.

Differentiation of enzyme and substrate binding in the prothrombinase complex.

The Ca2+ dependence of factor Xa binding to phospholipid vesicles was measured in the presence and absence of factor Va. The increase in polarization of a fluorescently labeled derivative of factor Xa, [5-(dimethylamino)-1-naphthalenesulfonyl] glutamylglycylarginyl factor Xa (Dns-EGR-Xa), was used as a probe to measure the interaction of factor Xa with phospholipid. The Ca2+ concentration required for half-maximal binding of Dns-EGR-Xa to phospholipid vesicles was 3.5 X 10(-4) M in the presence of factor Va and 9.5 X 10(-4) M in the absence of factor Va. At a Ca2+ concentration of 5 X 10(-4) M, the binding of Dns-EGR-Xa to phospholipid-bound factor Va was near maximal, whereas there was no detectable interaction of Dns-EGR-Xa with phospholipid alone at this Ca2+ concentration as detected by fluorescence polarization. These results were qualitatively confirmed by high-performance liquid chromatography. The rate of hydrolysis of the factor Xa synthetic substrate, benzoylisoleucylglutamylglycylarginine p-nitroanilide, by factor Xa in the presence of factor Va and phospholipid decreased in a Ca2+-dependent manner. These data were analyzed as fraction of factor Xa bound to the phospholipid. A Ca2+ concentration of 2.7 X 10(-4) M resulted in half-maximal binding by this technique. The relationship observed between rates of prothrombin activation and Ca2+ concentration could be predicted quantitatively from calculations of local enzyme and substrate concentrations.

A Modified Two-Stage Assay Method for Tissue-Thromboplastin (TPLN)

SummaryA two-stage method employing purified clotting factors for the assay of tissue-TPLN is described in detail. The rate of prothrombin activation is determined in the presence of optimum amounts of factors V, VII, X and Ca++. The activation of prothrombin is arrested by addition of EDTA or STI, and the thrombin formed is assayed by the clotting method. The method is rapid, sensitive, reproducible and can differentiate between the tissue-TPLN activity of intact lipo-protein and the procoagulant activity of the phospholipids.

Prothrombin Levels in Serum

SummaryProthrombin has been studied in clotting blood and plasma using the modified 2-stage prothrombin assay of Ware and Seegers. The quantity as well as the rate of prothrombin activation has been determined in whole blood and recalcified plasma obtained from 20 normal individuals. A thorough evaluation was made of the techniques used and the effect of variables such as temperature, surface contact, calcium, platelet concentration, and pH.A relatively constant rate of prothrombin activation was observed in clotting blood and recalcified frozen-thawed plasma.The results of this study confirm previous investigations of Langdell, Graham, and Brinkhous using the prothrombin utilization assay. In normal blood, the plasma procoagulants are found to be present in relative excess in comparison to platelet Factor 3. The lack of available platelet factor contained in the intact platelets effectively limits prothrombin activation. Disruption or alteration of the platelets permits the release of large quantities of platelet Factor 3 and produces a marked increase in the rate of prothrombin activation.Glass contact was observed to increase prothrombin activation in blood and plasma.The use of plasma prothrombin as a substrate for evaluation of procoagulant activity is discussed and advantages of this system of assay are emphasized.