Hans R. Baumgartner

The role of blood flow in platelet adhesion, fibrin deposition, and formation of mural thrombi.

Abstract Rabbit aortas were selectively denuded of endothelium by a balloon catheter, thus exposing the subendothelial surface. The interaction of platelets with this surface was examined by light and electron microscopy and measured with a morphometric technique. At unaltered flow in vivo platelets formed a tight and continuous layer within 10–20 min, no fibrin deposition was found, and formation of platelet thrombi was transient. A similar pattern of platelet interaction with subendothelium was observed in vitro after exposure to citrated blood which was pumped through a newly developed perfusion chamber at an average flow velocity similar to that measured in vivo . In contrast, at reduced blood flow in vivo markedly fewer platelets adhered to the subendothelial surface within a given period of time, areas with fibrin deposits at the subendothelial surface and in close association with platelets and red blood cells were observed and mural platelet thrombi were virtually absent. Similarly, fewer platelets adhered to the subendothelial surface at lower blood flow velocities in vitro and no platelet thrombi formed. These experimental data indicate that platelet adhesion and aggregation play a predominant role only at high, i.e., arterial, blood flow velocities and function independent of coagulation. Fibrin deposition at the subendothelial surface on the other hand appears to require stagnant blood.

Decreased adhesion of giant (Bernard-Soulier) platelets to subendothelium: Further implications on the role of the von Willebrand factor in hemostasis

Abstract The platelets of two patients with the Bernard-Souller (giant platelet) syndrome were not aggregated by bovine factor VIII. Platelet aggregation by rlstocetin was also absent and, in contrast to the findings in von Willebrands disease, this defect was not corrected by human factor VIII. The platelets of patients with the Bernard-Soulier syndrome may lack a receptor for the von Willebrand factor activity of factor VIII (VIII VWF ), whereas the abnormal platelet function in von Willebrands disease is due to a decreased level of VIII VWF in their plasma. As in the latter disorder, the adhesion of platelets to subendothellum was impalred in the two patients with the Bernard-Souller syndrome that we studied. These findings provide further evidence that VIII VWF , through its effect on platelets, plays an important role during the primary arrest of bleeding.

Adhesion of platelets to subendothelium.

Endothelium provides the physiologic nonthrombogenic surface in vivo. Connective tissue components underneath the endothelial lining form the subendo‐thelium. The subendothelial surface appears far less thrombogenic than collagen fibers. Platelet adhesion to subendothelium as an entity is greatly enhanced by the presence of other blood cells, by an increase in the flow rate of the circulating blood, and by the presence of divalent cations. The affinity of platelets to different connective tissue components of the subendothelium varies markedly. When exposed to the same blood stream in a newly developed perfusion chamber, the affinity of platelets to the respective material decreases in the following order: collagen fibers, amorphous material (basement membrane), microfibrils, and elastin.

Platelet interaction with subendothelium in a perfusion system: Physical role of red blood cells

Abstract Subendothelium from everted rabbit aorta was exposed in an annular perfusion chamber to whole blood and platelet rich plasma under controlled flow conditions. Platelet rich plasma was perfused in the in vitro system for periods as long as 100 min without altering the ability of the platelets to adhere to subendothelium. The rate of platelet deposition obtained from whole blood was approximately 57 times that from platelet rich plasma. Deposition results were compared with predictions obtained from mass transport theory. The analysis indicated that the increase in deposition caused by red cells was consistent with a physical, rather than humoral, mechanism—red cell motions in flowing blood increase platelet diffusivity by two orders of magnitude over that in plasma. The possibility of a minor humoral role for the red cells could not be entirely excluded.

Platelet interaction with subendothelium in flowing rabbit blood: effect of blood shear rate.

Abstract An annular perfusion chamber was utilized to expose subendothelium from everted rabbit aorta to blood flowing under controlled conditions. Initial platelet contact (C), platelet spreading (S), and the formation of platelet microthrombi (T) were evaluated by a morphometric technique for exposure times ranging from 1 to 40 min and flow rates of 10, 20, 40, and 160 ml/min (comparable to vessel wall shear rates of 650, 1300, 2600, and 10,000 sec−1). Analysis of the platelet adhesion results (C + S) with available mass transport theory indicated that, under these shear conditions, both platelet transport to the vicinity of the subendothelium and platelet reactivity with the vessel wall influenced the platelet attachment rate. At the highest shear rate studied (10,000 sec−1) an inhibition of platelet adhesion was observed which may be due to the effect of shear on either platelet transport or reactivity. The rate of formation of platelet microthrombi increased sharply between shear rates of 650 and 1300 sec−1 and continued to increase up to 10,000 sec−1 even though platelet adhesion was inhibited at this shear rate. Below 1000 sec−1 thrombi were always transiently formed, disappearing after exposure times of 20 min. Above 1000 sec−1 platelet thrombi formed from the blood of certain experimental animals had not disappeared at exposures as long as 40 min. The mechanisms governing the inhibition of platelet adhesion and the stabilization of platelet thrombi by shear rates encountered in certain areas of the microcirculation are currently unknown.

Shear Rate Dependent Inhibition of Platelet Adhesion and Aggregation on Collagenous Surfaces by Antibodies to Human Factor VIII/von Willebrand Factor

The effect of heterologous and homologous antibodies to factor VIII/von Willebrand factor (F.VIII/VWF) and purified VWF on the interaction of human platelets with subendothelium and with a surface consisting of collagen fibrils was investigated using annular flow chambers. Wall shear rates of 200–5200 s−1 were produced by varying the flow rate of the citrated blood which was circulated through these chambers by a pump. Surface coverage with platelets and with platelet aggregates was measured morphometrically. The antibodies had no effect on platelet adhesion at low shear rates. However, an increasing inhibition of adhesion was observed with increasing shear rates. The antibodies to F.VIII/VWF and to purified VWF virtually abolished adhesion at shear rates which are present in the microvasculature (1300–5200 s−1). By contrast, antibodies to factor VIII:C which were isolated from two patients with haemophilia A had no effect on adhesion. In addition antibodies to F.VIII/VWF and to purified VWF inhibited adhesion‐induced aggregation. We conclude that F.VIII/VWF plays an essential role as cofactor for platelet adhesion to subendothelium and collagen fibrils at high blood shear rates, i.e. when the time available for the establishment of a stable bond between platelets and collagenous surfaces is short.

Impaired interaction (adhesion-aggregation) of platelets with the subendothelium in storage-pool disease and after aspirin ingestion. A comparison with von Willebrand's disease.

Possible defects in the interaction of platelets with the subendothelial surface were evaluated in six patients with storeage-pool disease, nine patients with von Willebrands disease and seven normal subjects who ingested aspirin. Citrated blood was perfused through a chamber containing everted segments of rabbit aorta previously denuded of endothelium by means of a ballon catheter. With normal blood, 83.3 +/- 1.9 per cent (S.E.M.) of the surface was covered by adherent platelets. Platelet adhesion was normal after aspirin ingestion (89.7 +/- 4.6 per cent) and decreased in some patients with storage-pool disease. The most striking defect in both circumstances was the virtual absence of platelet thrombi. In contrast, decreased adhesion (57.3 +/- 3.4 per cent), but normal thrombus formation, was characteristic of von Willebrands disease. These types of defects in platelet adhesion and aggregation may account for the hemostatic defects in a variety of bleeding disorders. The findings further suggest the possible usefulness of aspirin as an antithrombotic agent.

The Proliferative Response to Vascular Injury Is Suppressed by Angiotensin-Converting Enzyme Inhibition

Smooth muscle cell (SMC) proliferation and formation of extracellular matrix in the intima of muscular arteries are major processes that can lead to vascular stenosis in arteriosclerosis or after coronary angioplasty. These processes are also seen in the proliferative response to balloon catheter-induced vascular injury of the rat carotid artery, and result in marked neointima formation by 14 days after catheterization. We have shown recently that the angiotensin-converting enzyme (ACE) inhibitor cilazapril strongly suppressed this development of neointima. In this report, we show that the beneficial effects on neointima formation persist for at least 8 weeks after stopping treatment with cilazapril, and that continuous treatment may have additional inhibitory effects during the late phases of vascular remodeling after injury. To investigate further the possible mechanisms, we examined several vasoactive compounds in this model. Another ACE inhibitor of a different chemical class, captopril, reduced neointima formation as strongly as cilazapril (67 and 78%, respectively), but the calcium antagonist verapamil was not active as an inhibitor of neointima formation, despite similar lowering of blood pressure. Hydralazine and a new calcium antagonist, Ro 40-5967, partially suppressed neointima formation (36%, p less than 0.005 and 33%, p less than 0.05, respectively). In vitro, neither cilazapril nor its active metabolite, cilazaprilate, had any effect on SMC proliferation in response to serum or PDGF. To characterize further the role of angiotensin II (Ang II), we tested in cell culture the effects of Ang II and cilazaprilate on mRNA levels of several proteins potentially involved in regulating the SMC response.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen induced platelet aggregation: Requirement for tropocollagen multimers☆

Abstract Fibrillar collagen suspensions and neutral salt soluble collagen were investigated with regard to their platelet aggregating activity. The activity of fibrillar suspensions was highly dependent on the method of preparation and was observed to increase with the fineness of dispersion. Monodisperse tropocollagen was found not to aggregate platelets. However, aggregating activity developed after multimerizing the molecules by incubation at 37° C. At this stage no fibrils could be detected by measuring the opacity at 400 nm. It is proposed that the triple helix is an insufficient structure for promoting platelet aggregation; multimers of tropocollagen are required to induce this platelet reaction. Evidence for a close correlation between fibril precipitation and development of aggregating activity is presented. Treatment of soluble collagen with galactose oxidase was shown to interfere with multimerization and consequently to lead to a delayed development of aggregating activity. Enzyme treated and control collagen showed similar aggregating activities after multimerization at 37° C. Treatment of fibrillar collagen with galactose oxidase produced no loss in aggregating activity as compared to control collagen.

Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin.

Exposure of type III collagen coats on plastic cover slips in parallel-plate perfusion chambers to flowing nonanticoagulated human blood resulted in deposition of platelets and fibrin. Blood was drawn directly from an antecubital vein by an occlusive roller pump over the collagen coats in chambers having flow slits of different dimensions, so that wall shear rates of 100, 650, and 2600 s-1 were obtained at 10 ml/min. Coagulation was minimally activated during the passage of blood from the vein to the chamber as shown by fibrinopeptide A levels of 3.7 ng/ml after 5-minute perfusions. The surface coverage with platelets increased from 18% at 100 s-1 to 59% at 2600 s-1, and the corresponding thrombus volumes increased from 2 to 22 microns 3/microns 2, respectively. This contrasted with the coverage with fibrin on collagen, which decreased from 28% at 100 s-1 to 9% at 2600 s-1. Fibrin deposits on the thrombi covered 6% of the surface irrespective of the shear rate, indicating that some of the deposited platelets accelerated the deposition of fibrin. The type III collagen preparation did not activate factor XII and did not possess tissue factor activity, indicating that the surface itself was not procoagulant. However, a correlation between deposited leukocytes and surface coverage with fibrin was observed (r = 0.78, p less than 0.01), suggesting a role for these cells in the deposition of fibrin. The data demonstrate that thrombogenesis is triggered by pure type III collagen, although the deposition of fibrin is not initiated by the collagen itself but presumably by deposited leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)