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Background: Effective HIV treatment with antiretroviral therapy has prolonged survival and shifted causes of death to non-AIDS illnesses such as cardiovascular disease. We have shown that inflammation and HIV viral load associate with pro- and anticoagulant factor imbalances resulting in increased thrombin generation when mathematically modeled. We explore the hypothesis that factor compositional imbalance, corresponding to increased in silico thrombin generation, predicts mortality among HIV+ persons., Methods: In a nested case-control study of HIV+ individuals on continuous antiretroviral therapy in two large trials, we evaluated cases (any non-violent mortality, n = 114) and matched controls (n = 318). Thrombin generation in response to a tissue-factor initiator for each individual was calculated by a mathematical model incorporating levels of factors (F)II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor, and protein C (PC) measured at study entry to the trials. In silico thrombin generation metrics included clot time, maximum rate (MaxR), maximum level (MaxL), and area under the curve (AUC)., Results: Levels of antithrombin and PC decreased, while FV and FVIII were higher in cases vs controls. This resulted in a more procoagulant phenotype with increased MaxR, MaxL, and AUC in cases compared to controls ( P  <   0.05 for all)., Conclusions: Antithrombin, FV, FVIII, and PC were the major contributors to the increased thrombin generation associated with mortality risk. Our results suggest that mortality in HIV is associated with an increase in in silico thrombin generation via altered balance of pro- and anticoagulant factors, likely due to an inflammatory response signal, and resulting coagulopathy.

Whole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5 gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient's plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor-initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient's platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence., (© 2015 by The American Society of Hematology.)

Objective: Normal pregnancy results in a prothrombotic state. Studies that have investigated the capacity of pregnant women to generate thrombin are limited. Our aim was to evaluate thrombin generation longitudinally from the preconception period, through pregnancy, and after pregnancy., Study Design: We evaluated young, healthy nulligravid women (n = 20) at 4 time points and compared the data with 10 control women at 2 time points. Coagulation was initiated with tissue factor in contact pathway inhibited plasma, and thrombin generation was determined in the presence of a fluorogenic substrate., Results: The maximum level and rate of thrombin generation increased during pregnancy; the highest level and rate occurred in late pregnancy compared with prepregnancy (P < .001). Subsequently, thrombin generation decreased in the postpregnancy samples that included maximum level, rate, and area under the curve (P < .001)., Conclusion: Our data provide evidence for an increase in tissue factor-dependent thrombin generation with pregnancy progression, followed by a return to prepregnancy thrombin levels., (Copyright © 2012. Published by Mosby, Inc.)

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R(506) and R(306). Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R(306) was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (+/-1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)- coupled microspheres and probed with mouse anti-human Ig-R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 +/- 0.8 nM), 11 BU/mL (214.3 +/- 7.1 nM), 106 BU/mL (2209.4 +/- 84.9 nM), 140 BU/mL (2417.7 +/- 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti-human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

We evaluated systemic alterations to the blood coagulation system that occur during a coronary thrombotic event. Peripheral blood coagulation in patients with acute coronary thrombosis was compared with that in people with stable coronary artery disease (CAD). Blood coagulation and platelet activation at the microvascular injury site were assessed using immunochemistry in 28 non-anticoagulated patients with acute myocardial infarction (AMI) versus 28 stable CAD patients matched for age, sex, risk factors, and medications. AMI was associated with increased maximum rates of thrombin-antithrombin complex generation (by 93.8%; P< .001), thrombin B-chain formation (by 57.1%; P< .001), prothrombin consumption (by 27.9%; P= .012), fibrinogen consumption (by 27.0%; P= .02), factor (f) Va light chain generation (by 44.2%; P= .003), and accelerated fVa inactivation (by 76.1%; P< .001), and with enhanced release of platelet-derived soluble CD40 ligand (by 44.4%; P< .001). FVa heavy chain availability was similar in both groups because of enhanced formation and activated protein C (APC)-mediated destruction. The velocity of coagulant reactions in AMI patients showed positive correlations with interleukin-6. Heparin treatment led to dampening of coagulant reactions with profiles similar to those for stable CAD. AMI-induced systemic activation of blood coagulation markedly modifies the pattern of coagulant reactions at the site of injury in peripheral vessels compared with that in stable CAD patients.

Aspirin is effective in the prevention of cardiovascular events in high-risk patients. The primary established effect of aspirin on hemostasis is to impair platelet aggregation via inhibition of platelet thromboxane A(2) synthesis, thus reducing thrombus formation on the surface of the damaged arterial wall. Growing evidence also indicates that aspirin exerts additional antithrombotic effects, which appear to some extent unrelated to platelet thromboxane A(2) production. Aspirin can reduce thrombin generation with the subsequent attenuation of thrombin-mediated coagulant reactions such as factor XIII activation. Aspirin also acetylates lysine residues in fibrinogen resulting in increased fibrin clot permeability and enhanced clot lysis as well as directly promoting fibrinolysis with high-dose aspirin. The variable effectiveness of aspirin in terms of clinical outcomes and laboratory findings, which has been termed aspirin resistance, may be related to these additional antithrombotic effects that are altered when associated with common genetic polymorphisms such as the Leu33Pro beta(3)-integrin or Val34Leu factor XIII mutations. However, the clinical relevance of these observations is still unclear. Elucidation of the actual impacts of aspirin other than antiaggregation effects could be important in view of the widespread use of this drug in the prevention of thrombotic manifestations of atherosclerosis.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.