Procoagulant Activity In Response Research Articles

OC 22.3 Monocytes and Macrophages Promote Increased Tissue Factor/Factor VIIa-Dependent Procoagulant Activity in Response to the SARS-CoV-2 Spike Protein

OC 22.3 Monocytes and Macrophages Promote Increased Tissue Factor/Factor VIIa-Dependent Procoagulant Activity in Response to the SARS-CoV-2 Spike Protein

57-Year-Old Woman With Weakness and Word-Finding Difficulties

57-Year-Old Woman With Weakness and Word-Finding Difficulties

Effects of antiplatelet therapy on platelet extracellular vesicle release and procoagulant activity in health and in cardiovascular disease

Dual antiplatelet therapy with aspirin and clopidogrel is commonly used to prevent recurrent ischemic events in patients with cardiovascular disease. Whilst their effects on platelet reactivity are well documented, it is unclear, however, whether antiplatelet therapy inhibits platelet extracellular vesicle (EV) release. The aim of this study was to investigate the effects of antiplatelet therapy on platelet EV formation and procoagulant activity.Blood samples from 10 healthy controls not receiving antiplatelet therapy were incubated in vitro with aspirin or a P2Y12 inhibitor (MeSAMP). Blood samples from 50 patients receiving long-term dual antiplatelet therapy and undergoing coronary angiography were also studied. Platelet reactivity was assessed by Multiplate™ impedance aggregometry. Platelet EV formation and procoagulant activity of pretreated and untreated blood samples in response to arachidonic acid (AA), adenosine diphosphate (ADP), ADP+PGE1, and thrombin receptor-activating peptide (TRAP) stimulation were assessed by flow cytometry and Procoag-PL assays, respectively.Incubation of normal platelets with aspirin significantly inhibited AA-induced platelet reactivity, EV formation, and procoagulant activity, whilst MeSAMP significantly inhibited platelet reactivity and EV formation in response to AA, ADP, and TRAP, but had minimal effect on procoagulant activity. Most patients receiving dual antiplatelet therapy showed an appropriate reduction in platelet reactivity in response to their treatment; however there was not complete inhibition of increased platelet and EV procoagulant activity in response to ADP, AA, or TRAP. In addition, we could not find any correlation between platelet reactivity and procoagulant activity in patients receiving dual antiplatelet therapy.

Intra-alveolar tissue factor pathway inhibitor is not sufficient to block tissue factor procoagulant activity

The alveolar compartment in acute lung injury contains high levels of tissue factor (TF) procoagulant activity favoring fibrin deposition. We previously reported that the alveolar epithelium can release TF procoagulant activity in response to a proinflammatory stimulus. To test the hypothesis that the alveolar epithelium further modulates intra-alveolar fibrin deposition through secretion of an endogenous inhibitor to TF, tissue factor pathway inhibitor (TFPI), we measured TFPI levels in edema fluid (EF) from patients with acute respiratory distress syndrome. To determine whether the alveolar epithelium can release TFPI, both full-length TFPI and truncated TFPI were measured (ELISA) in pulmonary edema fluid from patients with acute respiratory distress syndrome (ARDS) and a control group of patients with hydrostatic pulmonary edema (HYDRO). TFPI protein was also measured in conditioned media (CM) and cell lysates (CL) from human alveolar epithelial cells (A549) after exposure to cytomix (TNF-alpha, IL-1 beta, IFN-gamma). TFPI protein levels were higher in pulmonary edema fluid from patients with ARDS vs. HYDRO. TFPI protein was increased in CM and did not change in CL after cytomix treatment; TFPI mRNA levels (RT-PCR) did not change. Despite the high levels of TFPI, both the EF and CM retained significant TF procoagulant activity as measured by plasma recalcification time. The majority of intra-alveolar TFPI was in a truncated, inactive form, whereas the majority of TFPI released from cells was full length, suggesting different mechanisms of inactivation. In summary, the alveolar epithelium releases TFPI in response to an inflammatory stimulus but does not increase TFPI gene transcription or protein production. Levels of intra-alveolar TFPI in ARDS are not sufficient to block intra-alveolar TF procoagulant activity due to truncation and inactivation of intra-alveolar TFPI.

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen

Introduction Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role. Material and methods Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation. Results Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced. Conclusions Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3 h in PBG (PBS containing 0.5% BSA and 5 mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5 h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01 μg/ml or greater, and by zymosan concentrations of 0.1 μg/ml or greater. ROS production in response to incubation with PMA was signficantly increased starting at 10 −7 M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10 −7 M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3 h in RPMI with LPS (0.1 μg/ml), zymosan (10 μg/ml), PGN (10 μg/ml), and PMA (10 −7 M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

AbstractThe role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages.

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.

Coexpression offlt-1,flt-4 andKDRin Freshly Isolated and Cultured Human Endothelial Cells

Vascular endothelial cell growth factors (VEGF) are key modulators of endothelial cell growth and function. The class III receptor tyrosine kinases KDR and Flt-1 are high-affinity receptors for VEGF, while Flt-4 is a receptor for the recently identified VEGF-C. We have examined the expression offlt-1,flt-4 andKDRin human microvascular and large vessel endothelial cells and in a variety of other cell typesin vitro.Endothelial cells proliferated and exhibited increased procoagulant activity in response to VEGF.Flt-1,flt-4 andKDRwere detected in both freshly isolated endothelial cells, and in sparse and confluent endothelial cell cultures by RT-PCR. Attempts to modulate receptor expression by culturing cells at reduced oxygen tensions (2%) did not induce consistent changes inflt-1,flt-4 orKDRexpression. Incubation with tumor-conditioned medium or co-culture of endothelial cells with a range of breast and small cell lung carcinoma cell lines did not reproducibly alter receptor mRNA expression. However,flt-1,flt-4 andKDRtranscript levels were enhanced following treatment with tetradecanoylphorbol acetate.

Factors responsible for the differential procoagulant effects of diverse plasminogen activators in plasma

This study was designed to define the procoagulant effects of different plasminogen activators in recalcified plasma. Accordingly, a two-stage assay procedure was developed in which citrated plasma was recalcified and incubated for 1–7 min with streptokinase (SK), urokinase (UK), or tissue-plasminogen activator (t-PA). An aliquot of this first-stage plasma was added to second-stage plasma; procoagulant activity was measured as the acceleration of the clotting time compared with that in control first-stage plasma to which plasminogen activators had not been added. With this procedure, second-stage clotting times were significantly accelerated after activation of plasminogen compared with those in control plasma and were shortest with 1000 i.u./ml SK. The presence of increased thrombin activity in the recalcified citrated plasma was verified by measurement of increased concentrations of fibrinopeptide A, which were inhibited by addition of I U/ml hirudin or or U/ml heparin to plasma during incubation with the plasminogen activators. Activation of factor X was induced in plasma incubated with SK. The rate of activation of factor X was increased when thrombin was elaborated. Induction of procoagulant activity was found to be dependent on depletion of α 2-antiplasmin and on the presence of plasmin activity in plasma, and could be inhibited by aprotinin. Thus, procoagulant activity in response to pharmacologic activation of plasminogen is dependent on plasmin-mediated activation of factor X, and subsequent activation of prothrombin.

Mouse tumor-associated macrophages do not generate procoagulant activity in response to different stimuli.

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of human and experimental tumors, might contribute to fibrin deposition within malignant tissues through the production of procoagulant activity (PCA). We have studied the PCA of tumor-associated macrophages (TAM) in 2 poorly immunogenic, metastatic murine sarcomas (mFS6 and MN/MCA1); peritoneal macrophages (PM) from tumor-bearing and control animals were also studied, as reference cell populations. PCA was evaluated by a one-stage clotting assay immediately after isolation (basal PCA) and following in vitro stimulation. Basal PCA was very low (less than 1 U/10(4) macrophages) in all cell preparations. Exposure of PM from both normal and tumor-bearing animals to bacterial endotoxin (lipopolysaccharide, LPS), phorbol myristate acetate (PMA) or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in 10-, 7- and 3-fold increases in PCA, respectively. In contrast, TAM from mFS6 and MN/MCA1 consistently failed to generate PCA in response to different concentrations of the same stimuli. Treatment of TAM with aspirin did not affect cell unresponsiveness. Fluorescence microscopy showed that almost all PM were stained with fluorescein isothiocyanate (FITC)-LPS, while less than 10% of the TAM were stained. These data, coupled with previous evidence that TAM have a lower number of specific binding sites for phorbol esters than PM, suggest that the defective responsiveness of TAM to endotoxin, PMA and, possibly, FMLP, is due to the lack, or very low expression, of binding sites for these agents on the cell surface. The tumor environment may orient the functional status of in situ macrophages in a direction less favorable to the host.

Tissue factor induction in human monocytes. Two distinct mechanisms displayed by different alloantigen-responsive T cell clones.

One component of the cellular immune response to antigens is the expression of procoagulant activity (PCA) by monocytes and macrophages. Induction of human monocyte PCA in response to alloantigenic stimulation requires the collaboration of HLA-DR-responsive T cells. In mixed lymphocyte cultures (MLCs), the induction of monocyte tissue factor appears to be mediated exclusively by a T cell-derived lymphokine. We have used a soft agar cloning method to generate alloantigen-responsive T cell clones from MLCs between irradiated Daudi lymphoblastoid cells and human peripheral blood mononuclear cells. Developing clones were screened for the ability to induce PCA in fresh autologous monocytes in response to Daudi stimulator cells. PCA induction was observed with some, but not all, proliferating T cell clones and two modes of induction were apparent. Some T cell clones mediated PCA induction exclusively by lymphokine production, whereas other clones delivered induction signals by direct cellular collaboration with the monocyte effector cells. These two inductive pathways were represented in distinct, non-inclusive functional subsets of T cell clones. Constitutive production of soluble inducer signals was not observed in T inducer clones. The magnitude of the monocyte PCA response increased in response to an increase in the allogeneic stimulator/T clone responder ratio, and third-party allogeneic cells were unable to elicit the PCA-inducing lymphokine signals from T inducer clones. Both modes of induction were shown to generate tissue factor protein activity in monocytes. Collectively, these results suggest that PCA induction can be initiated in response to alloantigens through collaboration with certain OKT3+, OKT4+, OKT8-, OKM1- T inducer clones, and that induction can be mediated by at least two different functional subsets of human T cells. Stimulation with the appropriate alloantigen may elicit both lymphokine and T cell-contact pathways of induction of tissue factor in human monocytes.

A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

Microthrombosis during endotoxemia: Potential role of hepatic versus alveolar macrophages

Macrophages (Mφ) produce multiple inflammatory mediators which may contribute to the pathophysiological processes seen during sepsis. Since diffuse microthrombosis has been implicated as a potential etiology for organ dysfunction and failure in sepsis, the present study examined the production of procoagulant activity (PCA) by Mφ in response to endotoxin, characterized the activity, and evaluated methods to modify the response. Since hepatic and pulmonary dysfunction is a common complication of sepsis, rabbit Mφ were isolated from both pulmonary (A-Mφ) and hepatic (H-Mφ) sites. Both Mφ populations produced PCA in response to endotoxin in vitro. There is a rapid rise in activity with a peak at 20− to 30+ fold over background levels at 8 hr poststimulation. Although similar in their ability to enhance coagulation, the two Mφ PCAs were shown to differ markedly in other biochemical and functional assays. The A-Mφ PCA in contrast to the H-Mφ PCA, was resistant to heat inactivation, serine protease inhibition, and warfarin pretreatment, while indomethacin (a prostaglandin synthesis inhibitor) blocked A-Mφ PCA production but not the H-Mφ response. Corticosteroids totally blocked PCA production by both Mφ populations. Endotoxin, therefore, induces a rapid increase in Mφ PCA, and the magnitude and rapidity of the response argue for a potentially significant pathophysiologic role, in vivo. Although derived from a common progenitor, A-Mφ and H-Mφ produce a functionally discreet PCA. This differential response may partially explain the contradictory results obtained in studies using various cellular metabolism inhibitors, e.g., indomethacin and steroids. Therapeutic proposals must account for these potential selective sensitivities between various cellular populations.

Inhibition of platelet activation by prostacyclin: Possible consequences in coagulation and anticoagulation

In human blood and platelet rich plasma prostacyclin reduces or completely reverses the shortening of the recalcification time and Russell Viper venom time induced by kaolin or collagen. These results indicate that prostacyclin prevents development of platelet factor three activity by kaolin and collagen and possibly the development of additional procoagulant activity in response to collagen. Indomethacin and prostaglandin F 2α were without effect. In addition, prostacyclin, by preventing platelet activation, potentiates the anticoagulant effect of heparin.

Lymphoid procoagulant activity and mitogenesis in the C3H/HeJ mouse: discordant response to lipopolysaccharide stimulation.

Abstract Lymphoid cells from the C3H/HeJ mouse respond abnormally to bacterial lipopolysaccharide (LPS) in a number of in vitro functions. Abnormalities in both macrophage and lymphocyte populations have been suggested. In the present study, splenic mononuclear cells were isolated from C3H/HeJ, C3H/St, A/J, and BALB/c mice, and analyzed for mitogenesis and induction of procoagulant activity (PCA) by LPS prepared by either phenol or butanol extraction methods. All strains responded vigorously with resultant increased 3H thymidine uptake to Con A and PHA, and all but the C3H/HeJ responded well to butanol or phenol extracted LPS. However, the C3H/HeJ was unresponsive to phenol-extracted LPS and exhibited a poor but significant response to butanol-extracted LPS. In contrast, lymphoid cells from all strains, including the C3H/HeJ, exhibited a marked and equal increase of PCA in response to both phenol- and butanol-extracted LPS by using both splenic and peripheral blood lymphoid cells. The results obtained indicate that although the C3H/HeJ mouse may be unresponsive to a number of LPS-stimulated functions, there is no associated defect in PCA generation, a lymphocyte collaboration-dependent event.

Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl(2), the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl(2). To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl(2) on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl(2). When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl(2), the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl(2), the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl(2). When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl(2), the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl(2) was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant activity peak protein contained bands of 195,000, 148,000-120,000, 79,000, 61,000, 51,000, and 18,000 daltons whereas the pattern for the reduced thrombin-activated procoagulant activity peak protein always lacked the higher molecular weight bands, but consistently showed the four lower molecular weight bands to be well resolved. Taken together, these results imply that thrombin generates the FVIII procoagulant activity that is stabilized by 0.25 M CaCl(2) and elutes aberrantly from 4% agarose in that solvent.