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Biomedical subjects

B A Bouchard

Publications and source records attributed to B A Bouchard.

11 recordsLinked to original sources
PubMed2005

Endocytosis of plasma-derived factor V by megakaryocytes occurs via a clathrin-dependent, specific membrane binding event.

Megakaryocytes were analyzed for their ability to endocytose factor V to define the cellular mechanisms regulating this process. In contrast to fibrinogen, factor V was endocytosed by megakaryocytes derived from CD34(+) cells or megakaryocyte-like cell lines, but not by platelets. CD41(+)ex vivo-derived megakaryocytes endocytosed factor V, as did subpopulations of the megakaryocyte-like cells MEG-01, and CMK. Similar observations were made for fibrinogen. Phorbol diester-induced megakaryocytic differentiation of the cell lines resulted in a substantial increase in endocytosis of both proteins as compared to untreated cells that did not merely reflect their disparate plasma concentrations. Factor IX, which does not associate with platelets or megakaryocytes, was not endocytosed by any of the cells examined. Endocytosis of factor V by megakaryocytes proceeds through a specific and independent mechanism as CHRF-288 cells endocytosed fibrinogen but not factor V, and the presence of other plasma proteins had no effect on the endocytosis of factor V by MEG-01 cells. Furthermore, as the endocytosis of factor V was also demonstrated to occur through a clathrin-dependent mechanism, these combined data demonstrate that endocytosis of factor V by megakaryocytes occurs via a specific, independent, and most probably receptor-mediated, event.

Cell Differentiation↗
PubMed2003

How factor VIIa works in hemophilia.

The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and "acquired" hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5x normal platelets (10 x 10(8)/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 x 10(8)/mL activated platelets (5x normal); (d) with TF, 10 nM rFVIIa and 3-5x normal nonactivated platelets (6-10 x 10(8)/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.

Blood Platelets↗
PubMed2001

Platelets, leukocytes, and coagulation.

Considerable data now support the hypothesis that platelets actively regulate the propagation of coagulation by (1) expressing specific, high-affinity receptors for coagulation proteases, zymogens, and cofactors; (2) protecting the bound coagulation enzymes from inactivation/inhibition; (3) restricting coagulant activity to the site of vascular injury; and (4) amplifying the initiating stimulus to lead to explosive thrombin generation. Thrombin generation is sustained at the site of vascular injury by the recruitment of circulating monocytes and neutrophils to the growing thrombus via the interaction of PSGL-1, which is constitutively expressed by leukocytes, with P-selectin, which is expressed by activated platelets. Unique among cells, monocytes can provide the appropriate membrane surface for the assembly and function of all the coagulation complexes required for tissue factor-initiated thrombin production. More studies are required to further delineate the roles of neutrophils and lymphocytes in the procoagulant response. This review will discuss the recent investigations and controversies regarding the various mechanisms by which platelets and leukocytes function in, and regulate, thrombin generation.

Animals↗
PubMed2001

Platelet regulation of thrombin generation in cardiovascular disease.

Platelets are intimately involved in the events leading to cardiac ischemia through their release of bioactive substances, aggregation, and support of procoagulant reactions at sites of atherosclerotic plaque formation and rupture. This review article will focus on what is currently known about the regulation of thrombin generation on the surface of activated platelets, and how it relates to thrombus formation.

Arteriosclerosis↗
PubMed1999

Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.

Amino Acid Sequence↗
PubMed1997

Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation.

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.

Blood Platelets↗
PubMed1997

Human brain pericytes differentially regulate expression of procoagulant enzyme complexes comprising the extrinsic pathway of blood coagulation.

After vascular injury, pericytes may function in blood coagulation events that lead to thrombin formation due to their subendothelial location in the microvasculature. Pericytes from human cerebral cortex microvessels were isolated and characterized, and their ability to express and regulate procoagulant enzyme complexes was determined. Tissue factor was detected on the cell surface of cultured human brain pericytes by immunocytochemistry and was shown to form a functional complex with factor (F) VIIa to effect both FIX and FX activation. Treatment of pericytes with the calcium ionophore A23187 increased the observed tissue factor activity twofold to fivefold, which was shown to be due to an enhancement of cofactor activity and not the release of endogenous antigen stores. Pericytes also provided the appropriate membrane surface required for the assembly of a functional prothrombinase complex, so that in the presence of FVa and FXa, they effected thrombin formation 50 to 100 times faster than any other cell examined to date. In marked contrast to observations in other cell systems, pericyte expression of prothrombinase activity remained unaltered after treatment with A23187. As has been shown for platelets, the membrane receptor on pericytes for FXa assembly into the prothrombinase complex appears to at least partially consist of the FXa receptor effector cell protease receptor-1. These combined data indicate that pericytes can activate and propagate the coagulant response through the extrinsic pathway and that the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation. These observations support the concept that pericytes may play an important role in regulating coagulation events after cerebrovascular injury.

Blood Coagulation↗
PubMed1994

alpha-Thrombin-induced human platelet activation results solely from formation of a specific enzyme-substrate complex.

Prior studies using the mutant thrombin, thrombin Quick I, indicate that this protease induces maximum platelet aggregation and intraplatelet [Ca2+] fluxes at agonist concentrations where dissociable, equilibrium platelet binding is undetectable and led to the conclusion that thrombin interaction with its platelet "receptor" is best described kinetically by formation of an enzyme-substrate complex. This conclusion was substantiated further in the present studies by demonstrating that both thrombin Quick I and thrombin mimicked the thrombin receptor agonist peptide in the induction of the platelet activation-dependent events required for functional Prothrombinase assembly and that a rabbit antibody raised against KATNATLDPRSFLLR, a pentadecapeptide which represents amino acids 32-46 in the platelet thrombin receptor/substrate and spans the thrombin cleavage site, inhibited both thrombin- and thrombin Quick I-induced platelet activation responses equivalently. The antipeptide antibody had a more pronounced inhibitory effect on the rate of the thrombin-induced response rather than the magnitude of the response suggesting competition for the cleavage site, consistent with the observation that pretreatment of metabolically-inhibited platelets with thrombin, which was removed by washing, eliminated specific antibody binding due to removal and/or masking of antibody epitopes. Concentrations of the antipeptide antibody that inhibited thrombin- and thrombin Quick I-induced increases in intracellular [Ca2+] flux by as much as 97% did not alter the dissociable equilibrium binding of 125-I-FPR-thrombin to platelets. These combined data indicate that the hydrolytic event initiated by thrombin or thrombin Quick I interaction with the platelet receptor/substrate for thrombin is unrelated to the dissociable equilibrium binding of thrombin to membrane sites described previously by classical receptor-ligand binding analyses.

Amino Acid Sequence↗