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

Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Relative involvement of GPIb/IX-vWF axis and GPIIb/IIIa in thrombus growth at high shear rates in the guinea pig.

The relative involvement of the glycoprotein (GP) Ib/IX-von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s(-1), in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s(-1), collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 microm3/microm2, respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.

Guinea pig blood: a model for the pharmacologic modulation of the GPIb/IX-vWF axis.

Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed in an ex vivo capillary perfusion chamber exposing human type III collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s-1) and for two perfusion times (1.5 and 4 min). At 1800 s-1 and 4 min of perfusion, platelet adhesion decreased from 63 +/- 7% for control, to 46 +/- 4% for 20 mg/kg RG12986, and to 29 +/- 5% for 4 mg/kg VCL, and the mean thrombus height dropped from 40 +/- 8 microns to 24 +/- 3 microns and 7.5 +/- 1 microns, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors.

Optimal antagonism of GPIIb/IIIa favors platelet adhesion by inhibiting thrombus growth. An ex vivo capillary perfusion chamber study in the guinea pig.

To evaluate the involvement of the glycoprotein (GP) IIb/IIIa-dependent process in platelet deposition and thrombus growth on capillaries coated with human type III collagen, the effects of incremental doses of Lamifiban, a potent specific synthetic GPIIb/IIIa antagonist, were studied in ex vivo capillary perfusion chambers using guinea pig blood. In this model, nonanticoagulated blood was perfused for 4.5 minutes at three shear rates: 100, 650, and 1600 s-1. Platelet deposition was quantified by computer-assisted morphometry and expressed as platelet adhesion (percentage of capillary surface covered with spread and contact platelets and platelets implicated in thrombus), mean thrombus height, and total thrombus cross-sectional area. In control untreated guinea pigs, platelet adhesion and thrombus height were 63% and 2.5 microns at 100 s-1, 60.5% and 13.8 microns at 650 s-1, and 45% and 28.1 microns at 1600 s-1, respectively. At 100 s-1, Lamifiban had no effect on platelet deposition at any of the three doses administered to the guinea pigs (0.3, 1, and 3 mg/kg). At 0.3 mg/kg and shear rates of 650 and 1600 s-1, Lamifiban had no effect on platelet adhesion or thrombus size, but at 1 and 3 mg/kg and shear rates of 650 and 1600 s-1, it significantly reduced thrombus size. At 1600 s-1, 1 mg/kg Lamifiban significantly increased platelet adhesion from 45% to 62.5%, whereas at 3 mg/kg it induced a significant overall decrease from 45% to 25% and qualitatively increased the ratio of contact to spread platelets. These data suggest that at high shear rates, GPIIb/IIIa participates in platelet spreading and that there is a balance between platelet involvement in adhesion to the thrombogenic surface and the growth of the already formed thrombus. This indicates that important clinical implications of an optimal therapeutic degree of GPIIb/IIIa antagonism could be expected.

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)

Reduction in thrombus formation by PG-1 F(ab')2, an anti-guinea pig platelet glycoprotein Ib monoclonal antibody.

We have recently demonstrated that intraperitoneal injection into guinea pigs of F(ab')2 fragments of PG-1, a murine monoclonal antibody recognizing the guinea pig homologue of human platelet glycoprotein Ib, produces virtually complete functional block of the platelet von Willebrand factor receptor without inducing a hemorrhagic state. To assess the ability of this antibody to protect against thrombosis resulting from laser-induced injury to mesenteric small arteries, we injected guinea pigs with either PG-1 F(ab')2 (2.3 mg/kg) or with buffer alone 24 hours before study. For each animal, four mesenteric vessels were studied consecutively for 15 minutes each. The number of thrombi, time to first thrombus, and time to embolization of first thrombi were recorded. In the control animals most individual vessels had three to four thrombi, whereas in the antibody-treated animals, vessels most frequently had only a single thrombus or even none at all. The mean number of thrombi per vessel in the antibody-treated animals (1.125) was significantly lower than the mean in the control animals (3.40), with p less than 0.001. However, there was no significant difference between groups with respect to time to first thrombus after laser injury. Detachment of thrombi in the control animals occurred in an easily recordable, discrete fashion, with 75% of first thrombi embolizing in less than 5 minutes. In the antibody-treated animals, gradual dissolution rather than discrete detachment was typically observed. In 31% of the vessels studied in these animals, no embolus occurred; for the remaining vessels only 9% of thrombi were observed to embolize within 5 minutes of their formation.(ABSTRACT TRUNCATED AT 250 WORDS)