Role of plasma and platelet von Willebrand factor in arterial thrombogenesis and hemostasis in the pig.

To evaluate the relative role of plasma and platelet von Willebrand factor (vWF) pools in hemostasis and arterial thrombogenesis, pigs with vW disease (vWD) were injected with vWF concentrate and/or grafted with bone marrow from a normal pig. Hemostasis was assessed by measurement of ear immersion bleeding time, factor VIII (FVIII) activity, and plasma and platelet vWF antigen levels. The thrombotic process was explored at 650 s(-1) and 1600 s(-1) in an ex vivo cylindrical perfusion chamber. Pigs with vWD exhibited a prolonged bleeding time (>30 minutes) compared with normal pigs (<5 minutes); in addition, they showed normal platelet adhesion and thrombus formation at 650 s(-1) but profoundly reduced platelet adhesion and thrombus formation at 1600 s(-1). Each experiment was performed before and 3 and 24 hours after injection of vWF concentrate. In our bleeding time study, only plasma vWF restoration induced a partial but delayed correction (24 hours postinjection), which was correlated with the highest measured level of FVIII activity. In the perfusion chamber model, restoration of plasma or platelet vWF pools resulted in similar partial correction of platelet adhesion and average thrombus size. In the perfused pigs, the maximum correction occurred 3 hours postinjection. Platelet deposition reached normal values after vWF concentrate was injected into a grafted pig. The present results suggest that when both plasma and platelet vWF levels are restored in vWD pigs, bleeding time and the thrombotic process are normalized according to different kinetics and with differing degrees of effectiveness.

An experimental model for testing von Willebrand factor function: successful SLA-matched crossed bone marrow transplantations between normal and von Willebrand pigs.

To assess the relative in vivo roles of von Willebrand factor (vWF) of different origins, we performed crossed bone marrow transplantations (BMTs) among normal pigs and pigs with the von Willebrand disease(vWF). The two groups were fully compatible immunologically according to typing by swine leukocyte antigen (SLA). After total-body irradiation (8-10 Gy), all pigs received 0.5X10(9) to 10(10)/kg mononuclear bone marrow cells without any immunosuppression. The nadir of aplasia occurred between days 5 and 7 after irradiation (white blood cell [WBC] count 0.6X10(9)/L, platelet [Plt] count 76X10(9)/L. Three weeks after the graft, WBC and Plt counts had returned to normal levels. Animals were followed for at lease 50 days, during which no bone marrow rejection occurred; no evidence of graft-vs-host disease (GVHD) was observed. Each BMT was confirmed by karyotype analysis. In the six homozygous pigs with vWD grafted with normal marrow, platelet vWF antigen (vWFAg) and platelet vWF activity rose from <3 to 450 U/dl with a normal multimeric pattern; plasma vF increases slightly. No correction of bleeding time was observed. In the five normal pigs grafted with bone marrow form pigs with vWD, platelet vWFAg and platelet vWF activity decreased from >100 U/dl to undetectable levels; bleeding time and plasma vWFAg remained unchanged. A derivative of normal porcine plasma, a concentrate containing factor VIII and vWF, was infused into a homozygous vWD pig before and after BMT from a normal pig. Co correction of bleeding time was obtained, even though plasma nd platelet vWFAg levels were normal. W concluded that crossed BMT among SLA-identical pigs is a feasible model of studying the synthesis and the roles of vWF in hemostasis and thrombosis.

The calcium inhibitor SR33805 reduces intimal formation following injury of the porcine carotid artery.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20

Histochemical and ultrastructural characterization of subendothelial glycoprotein microfibrils interacting with platelets.

The interaction of human blood platelets with collagenase-treated rabbit subendothelium was studied by histochemical ultrastructural methods and by morphometric semi-quantitative analysis. Aortas were deendothelialized and incubated: 1) with a highly purified bacterial collagenase whose specificity was controlled; and 2) with the same collagenase followed by chymotrypsin. For histochemical studies, tannic acid, ruthenium red, and peroxidase-labeled Ricinus communis and concanavalin A were used. Electron microscopy showed that after digestion of fibrillar collagen by collagenase, adherent and aggregated platelets were observed on Ricinus communis-, concanavalin A-, and ruthenium red-positive glycoprotein microfibrils. After successive incubation with collagenase and chymotrypsin, the microfibrils disappeared. No platelets were observed on the remnant amorphous elastin. Morphometric analysis confirmed the interaction of platelets with collagenase-treated subendothelium. In addition, glycoproteins were extracted from collagenase-treated rabbit aortas using 5 M guanidine. Using an in vitro quantitative test, significant platelet adhesion to these glycoproteins was observed. Our results show an interaction between platelets and noncollagenic glycoprotein microfibrils.

Comparative arterial antithrombotic activity of clopidogrel and acetyl salicylic acid in the pig.

We investigated the comparative antithrombotic properties of clopidogrel, an analogue of ticlopidine, and aspirin, using the Folts' model on femoral arteries in 22 pigs. On each animal, clopidogrel or aspirin were used to treat the thrombotic process on the left femoral artery and to prevent this process on the right femoral artery. Sequentially: an injury and stenosis were carried out on the left femoral artery; the thrombotic process was monitored with a Doppler during a 30-min observation period for cyclic flow reductions or permanent cessation of flow; after the first cyclic flow reduction occurred, clopidogrel (5 mg kg-1) or aspirin (2.5, 5, 100 mg kg-1) were injected intravenously; if cyclic flow reductions were abolished, epinephrine (0.4 micrograms kg-1 min-1) was injected to try to restore cyclic flow reductions and/or permanent cessation of flow; then injury and stenosis were applied on the right femoral artery. Before and after injection of clopidogrel or aspirin, ear immersion bleeding times and ex-vivo platelet aggregation were performed. Clopidogrel (n = 7) abolished cyclic flow reductions were efficiently prevented, even for two injuries. Basal bleeding time (5 min 28) was lengthened (> 15 min, 30 min after clopidogrel and remained prolonged even after 24 h). ADP-induced platelet aggregation was inhibited (more than 78%). Comparatively, aspirin had a moderate and no dose-dependent effect. Aspirin 2.5 mg kg-1 (n = 6) abolished cyclic flow reductions in 2 animals, CFR reoccurred spontaneously in one animal and epinephrine restored it in a second animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Aprotinin could promote arterial thrombosis in pigs: a prospective randomized, blind study.

Haemostatic properties of aprotinin could be associated with an increased risk of thrombosis. A randomized, blinded study was conducted to consider the potential thrombogenicity of aprotinin, using the Folts' model on femoral arteries in 12 pigs. The flow variations were measured by a pulsed Doppler in anaesthetised animals. Ear immersion bleeding time was performed. During the first part of the study, a stenosis was performed successively on both femoral arteries, each for a period of 30 min, without prior injury, to assess the integrity of the vessel, and to check that the arteries did not develop cyclic flow reductions (CFR), permanent cessation of flow (PCF) or partial thrombosis, when a stenosis is applied. Then the clamp was released and a bolus of placebo (saline), or aprotinin (4 millions KIU, followed by a continuous infusion of 1 million KIU.h-1), was administered. At the end of the bolus, the second part of the study began. Stenosis was applied to the arteries. If CRF, PCF, or partial thrombosis were observed without prior injury then the infused drug (aprotinin or saline) was considered a prothrombotic drug, and the opposite artery was studied. For each animal, right and left femoral artery segments were fixed and studied (morphologic study). Eighteen arteries were studied. In the aprotinin group, 6 arteries out of 8 developed an unexpected thrombosis, as compared with only 2 out of 10 arteries in the control group (p = 0.02). The morphologic study confirmed the occurrence of thrombosis in 4 out of 7 arteries in the aprotinin group, as compared with only 1 out of 9 in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Macro and microheterogeneity in normal endothelial cells: differential composition of luminal glycocalyx and functional implications.

Endothelial cells (EC) are involved in various physiological and pathological processes through the expression of their surface glycoproteins. They are covered by the glycocalyx, composed of glucidic residues from cell surface membrane glycoproteins, glycoplipids and proteoglycans. Glucidic sequences can be specifically characterized by their binding to lectins. Eight lectins were used to investigate the distribution and regulation of EC surface glucidic residues in various blood vessels of adult and newborn pigs. EC lectin binding was compared to von Willebrand factor (vWF) expression as EC reference marker. Six out of eight lectins (BSI-B4, DBA, EEA, HP, MAL I and PNA) were helpful for this determination. Considering only the intensity of labelings, vWF and DBA gave the best stainings of adult pig ECs. In newborn pigs, the best labelings were obtained with EEA and MAL I. Furthermore, the distribution of lectin binding to ECs and EC vWF expression was heterogeneous depending on the EC location along vascular tree and age. Beside this macroheterogeneity this study highlights a microheterogeneity of EC lectin binding and vWF expression in situ, defined as a staining of equal intensity by individual ECs, scattered among negative ones, in a given vascular segment. EC surface sugar residues were differently modulated in newborn and adult pig ECs and differently according to EC vWF expression. The functional involvement of EC glycocalyx was reflected by EC lectin binding in the spleen and liver. This study emphasizes the high level of EC heterogeneity for various markers. The EC macro- and microheterogeneity reflect the "plasticity" or "unstability" of EC phenotypes and suggests that ECs are subject to several levels of regulation and are probably grouped in functional clusters to best adjust their functions to microenvironmental requirements. This concept must be considered in further investigations notably in in vitro studies where EC phenotype can be altered.

Platelet functions and haemostasis parameters in pigs: absence of side effects of a procedure of general anaesthesia.

Pigs are largely used as experimental animal models of thrombosis and for testing the anti thrombotic drug efficacy. Generally experiments are performed on pigs under general anaesthesia and observations can be affected by the anaesthetic drugs used. The effects of a general anaesthetic procedure were checked on pig haemostasis parameters; the pig was pre-anaesthetized with ketamine chloride, then intubated and ventilated with a mixture containing halothane, nitrous oxide and oxygen. Bleeding time, platelet aggregations, coagulation factors, coagulation inhibitors, fibrinolysis parameters and markers of activation of coagulation were determined on 30 Large White pigs before and under this anaesthesia procedure. Compared to human coagulation, pig is characterized by very high levels of factor V, VIII, IX, XI, XII activities, same levels of factor II, fibrinogen, antithrombin III (ATIII), low levels of protein C activities. Thrombin-antithrombin complex (TAT) and tissue plasminogen activator antigen (tPA) values were dispersed. With the reagents used, protein S, prothrombin fragment 1 + 2 (F1 + 2), D Dimers (D-D), plasminogen activator inhibitor (PAi) levels could not be determined. No difference was observed between results obtained before and under anaesthesia, particularly to increase of bleeding time, no modification of platelet aggregations and no activation of coagulation. This anaesthetic procedure does not induce any modification of pig haemostasis and can be used, without side effects, for experimental thrombosis studies in pigs.

Clopidogrel-induced qualitative changes in thrombus formation correlate with stent patency in injured pig cervical arteries.

Thienopyridines (ticlopidine or clopidogrel) alone or in combination with aspirin are now the reference antiplatelet therapy after stent implantation. To better understand the high efficacy and low risk of bleeding with these agents, we tested clopidogrel alone or with aspirin in an acute ex vivo flow chamber model and in a subacute in vivo arterial thrombosis model. Clopidogrel induced a dose-dependent increase in bleeding time (BT), inhibited ADP-induced platelet aggregation and in the flow chamber reduced thrombus size, and changed thrombus structure to broad-based structure composed of nondegranulated loosely attached platelets contrasting with the tight clumps of degranulated platelets seen without clopidogrel. The in vivo model involved angioplasty and stenting at the site of a preinduced arterial lesion and thrombosis in pig carotid arteries. Clopidogrel alone or with aspirin (but not aspirin alone) decreased the number of stented vessels occluded for more than 24 h and conversely reduced the number of occluding thrombus. At 96 h after stenting, 100% and 90% of the arteries were patent with clopidogrel/aspirin and clopidogrel alone, respectively (vs. 67% and 44% with aspirin and saline, respectively). Clopidogrel destabilizes thrombus without complete abolishment of platelet reactivity.

Effect of kappa-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig.

An undecapeptide (residues 106-116 of cow kappa-casein) is known to inhibit human platelet aggregation and fibrinogen binding through inhibition of the interaction between the fibrinogen gamma-chain C-terminus and alphaIIbbeta3. This was due to structural homologies with the fibrinogen gamma-chain C-terminal dodecapeptide. We have therefore compared in this work the in vitro anti-aggregating activity of kappa-casein split peptides and their in vivo potential antithrombotic activity in a model of arterial thrombosis triggered by laser-induced intimal injury in the guinea-pig. Caseinoglycopeptide (residues 106-169), the undecapeptide (residues 106-116) and the pentapeptide KNQDK (residues 112-116) from cow kappa-casein, were anti-aggregating peptides and exerted a significant antithrombotic activity in the guinea-pig. Caseinoglycopeptides from three species (cow, ewe and human) were also antithrombotic and the most potent being the human one. The antithrombotic activity was achieved in vivo for doses less than the one suspected from in vitro data and for which, ex vivo platelet aggregation was not decreased. In conclusion, the relative involvement of the fibrinogen gamma-chain C-terminal dodecapeptide could be much more important in in vivo thrombosis process than in in vitro platelet aggregation. Its specificity and activity in vivo unveiled an interesting potential way for inhibition of arterial thrombosis if alternative molecular presentation (i.e. peptidomimetics) and alternative route (i.e. per os) can be developed.

Effects of human recombinant, plasma-derived and porcine von Willebrand factor in pigs with severe von Willebrand disease.

The effects of the infusion of a human recombinant von Willebrand factor (vWF) preparation in pigs homozygous for von Willebrand disease (vWD) were evaluated on serial measurements of von Willebrand factor antigen and activity, FVIII activity, vWF multimer analysis, in-vivo bleeding time and platelet adhesion and thrombus formation on collagen at high shear rates in an ex-vivo model of experimental thrombosis. Plasma-derived human and porcine vWF were used for comparison. Before infusion, the pigs were characterized by undetectable plasma vWF levels, a low level of FVIII, prolonged bleeding time, severely impaired platelet adhesion and thrombus formation. After infusion of the human recombinant vWF, in-vivo recovery of vWF activity ranged from 58% to 82%, depending on the dose infused, and its half-life was longer than for the plasma-derived concentrates. The highest-molecular-weight forms of human recombinant vWF were removed from the circulation gradually. Infusion of the three vWF concentrates produced inconsistent effects on bleeding time and moderate improvement of platelet adhesion and thrombus formation. After infusion, a prolonged increase of FVIII (> 48 h) was observed, suggesting that human recombinant vWF is able to bind and to stabilize porcine factor VIII and that porcine vWD is a good model for studying such interactions.

[What are the criteria for choosing a model for evaluating arterial antithrombotic therapy?]. / Sur quels critères choisir un modèle pour évaluer une thérapeutique antithrombotique artérielle?

The use of models of experimental arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step to the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost ...) which necessary lead to some promises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to choose a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts thus we have omitted their discussion from this paper.

Human blood platelet elastase and proelastase.

Platelet elastase has been differenciated from various protein fractions into a trypsin dependent form and a trypsin independent form. Trypsin independent elastase has been purified by affinity chromatography on cellulose elastin column as a pure protein raction of molecular weight: 26,000 ou SDS acrylamide gels. Trypsin dependent elastase has been purified by preparative acrylamide disc gel electrophoresis. This fraction, proteolysed (limited proteolysis) and activated by trypsin into active elastase, has been identified as the precursor (platelet proelastase) of platelet elastase. Its molecular weight is 28,000 before trypsin and 26,000 after trypsin.

Differentiation of the elastase-type protease of platelets from other elastases.

Elastase activities were determined near neutral pH on several specific substrates using platelet-derived preparations mixed with decreasing amounts of leukocytes. Activities were extrapolated to zero leukocyte content enabling the estimation of intrinsic platelet elastase activity. In contrast to human leukocyte elastase, metal chelating agents inhibited partly the elastase activity of the platelet extract and soybean trypsin inhibitor did not modify its activity. Serine active site titrants (phenylmethane sulfonyl fluoride) as well as acetyl-di-L-alanyl-L-propyl-L-valine chloromethylketone completely abolished the activity of platelet lysates. The platelet protease was purified from Triton X-100 platelet lysates. No cross-reactivity could be demonstrated by immunoelectrophoresis with either porcine pancreatic elastase or human leukocyte elastase using monospecific antisera. Applying gel electrophoresis, most of the elastase activity of the platelet protease migrated towards the anode, whereas the pancreatic and leukocyte elastases migrated towards the cathode. The anionic character of the platelet enzyme might explain its capacity to degrade better elastin treated with cationic detergents in contradistinction to other elastases which act better on anionic detergent-treated elastins.

Human blood platelet elastase and proelastase. Activation of proelastase and release of elastase after ahesion of platelets to collagen.

After an in vitro incubation of platelets with fibrillar collagen, their elastase activity is markedly and rapidly increased while proelastase decrease: proelastase is activated in situ into elastase which is released in its active form from the platelet. The activation of proelastase is likely due to the action of a trypsin-like enzyme present in the platelet. This protease has the same type of localization as proelastase and elastase: their highest activity is associated with light granules but part of these enzymes (or precursor) is also associated with the membranes. The mechanism of the arterial elastolysis induced by the platelets probably involves their adhesion to intimal thrombogenic surfaces (collagen) followed by a reaction during which proelastase would become available to the trypsin-like enzyme and would be activated into elastase directly released in the vessel wall.