Red blood cells: their dual role in thrombus formation.

Red blood cells may have a physical and chemical effect on the interaction between platelets and blood vessel surfaces. Under flow conditions in which primarily physical effects prevail, platelet adhesion increases fivefold as hematocrit values increase from 10 to 40 percent but undergoes no further increase from 40 to 70 percent, implying a saturation of the transport-enhancing capabilities of red cells. For flow conditions in which platelet-surface reactivity is more dominant, platelet adhesion and thrombus formation increase monotonically as hematocrit values increase from 10 to 70 percent. Thus red cells may have a significant influence on hemostasis and thrombosis; the nature of the effect is apparently related to the flow conditions.

Role of shear rate and platelets in promoting fibrin formation on rabbit subendothelium. Studies utilizing patients with quantitative and qualitative platelet defects.

The deposition of platelets on subendothelium of rabbit aortic segments exposed to non-anticoagulated human blood increased progressively with increasing wall shear rates (50-2,600 s-1), whereas fibrin deposition decreased. Studies in normal subjects and patients with platelet disorders suggested that, under the conditions used, platelets were essential for fibrin deposition at intermediate (650 s-1) but not low (50 s-1) shear rates. Fibrin deposition was markedly diminished in a patient with Scott syndrome whose platelets have a diminished capacity to bind Factor Xa and activate Factors IX and II. In glycoprotein IIb-IIIa deficiency, fibrin deposition was normal (or somewhat increased), whereas in glycoprotein Ib deficiency the association of fibrin with platelets, but not subendothelium, was decreased. The findings indicate that platelets, perhaps through surface localization of coagulation proteins, promote fibrin deposition on subendothelium at arterial shear rates and suggest that agents directed against platelet coagulant properties could be antithrombotic.

Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion.

Blood interaction with the subendothelium of rabbit aorta was investigated in an annular perfusion chamber using patients with von Willebrand's disease, hemophilia, and afibrinogenemia. The vessels were exposed to nonanticoagulated blood for a range of flow conditions (wall shear rates of 650-3,300 s-1) and exposure times (1.5-10 min). The resultant platelet and fibrin interaction was quantified by the use of several morphometric techniques, one of which was developed to measure more precisely the dimensions (height and volume) of platelet thrombi attached to the subendothelium. A major finding was that under flow conditions in which little or no defect in platelet adhesion was observed in von Willebrand's disease, platelet thrombus height and volume in this disorder were significantly reduced as compared with normal controls or patients with hemophilia. Thus, Factor VIII/von Willebrand factor (VIII/VWF) may mediate not only the adhesion of platelets to subendothelium but also platelet-platelet attachments necessary for normal thrombus development. The level of Factor VIII:coagulant activity (VIII: C) was also observed to influence the resultant thrombus height and volume deposited on subendothelium, presumably through the generation of thrombin or some other procoagulant factor preceding fibrin formation, since normal values of thrombus dimensions were always observed in a patient with a fibrinogen deficiency. The influence of VIII:C became greater as shear rate was reduced, whereas as shear rate was increased, VIII/VWF was more dominant in determining the resultant platelet deposition on subendothelium. Thus, the deficiencies of VIII:C and VIII/VWF in hemophilia and von Willebrand's disease can lead to various abnormalities in platelet and fibrin association with subendothelium. The importance of a particular deficiency will depend strongly on the local blood flow conditions.

Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate.

Platelet adhesion and thrombus formation on subendothelium, studied at a shear rate of 2,600 s-1, were inhibited by two synthetic peptides known to interact with GPIIb-IIIa. One peptide (HHLGGAKQAGDV) corresponds to the carboxyl terminal segment of the fibrinogen gamma-chain (gamma 400-411) and the other (RGDS) contains the amino acid sequence Arg-Gly-Asp (RGD) common to fibronectin, von Willebrand factor, vitronectin and the alpha-chain of fibrinogen. Neither platelet adhesion nor thrombus formation were decreased in a patient with severe congenital fibrinogen deficiency and this was equally true when his blood was further depleted of the small amounts of fibrinogen present utilizing an anti-fibrinogen antibody. In normal subjects, adhesion and thrombus formation were inhibited by the Fab' fragments of a monoclonal anti-GPIIb-IIIa antibody (LJ-CP8), which interferes with the interaction of platelets with all four adhesive proteins in both the fluid and solid phase. However, another anti-GPIIb-IIIa antibody (LJ-P5) that had minimal effects on the interaction of platelets with fibrinogen, but inhibited to varying degrees platelet interaction with other adhesive proteins, was equally effective. The findings demonstrate that, at a shear rate of 2,600 s-1, adhesive proteins other than fibrinogen are involved in GPIIb-IIIa-mediated platelet adhesion and thrombus formation on subendothelium. In addition, since LJ-P5 inhibited the binding of soluble von Willebrand factor and vitronectin, these adhesive proteins may be involved in platelet thrombus formation. In contrast to the results obtained at a shear rate of 2,600 s-1, fibrinogen could play a role in mediating platelet-platelet interactions with weak agonists or lower shear rates.

The effect of flow on hemostasis and thrombosis.

While dilution of procoagulants has generally been proposed as the mechanism by which flow reduces coagulation at surfaces, such a mechanism has never been verified experimentally and, in fact, there are theoretical grounds for suspecting the validity of such a hypothesis (29). It is quite plausible that flow may have direct effects on certain enzyme or polymerization kinetics involved in thrombosis, in addition to the well-defined effect that flow has an enhancing transport of reactants and products to and from the vessel wall. Such effects of flow on immobilized enzymes have occasionally been observed, but never studied with respect to coagulative processes (30). The study of the effects of flow on hemostasis and thrombosis, while numerous, are still in their infancy. As noted above, increasing shear increases the rate of formation of factor Xa in a tubular reactor. In the presence of factors VIII and IX, there is also a shear-induced enhancement of Xa production (31). These studies indicate that at least some coagulation reactions are accelerated in the presence of high shear. However, it has been observed that fibrin formation is diminished at increasing shear rates (20). This implies that at least one step of the coagulation cascade is being inhibited by high shear. One possibility is that fibrin monomer is being removed by the high local flow conditions, although the concomitant reduction in fibrinopeptide A argues against this interpretation. Another possibility, not yet tested, is that thrombin itself is removed by flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of quantifiable hemodynamic factors in the assessment of cerebral aneurysm behavior. On behalf of the Subcommittee on Biorheology of the Scientific and Standardization Committee of the ISTH.

Previous experimental and theoretical studies on the hemodynamics of saccular intracranial aneurysms have provided evidence that aneurysms tend to grow, thrombose and rupture when (1) wall shear stress and mural tension are increased compared to normal values, and (2) flow deviates from a laminar unidirectional pattern (for example flow recirculation). Aneurysm wall shear stress, however, is the only hemodynamic factor which has received special attention in terms of estimation. Additional flow-related parameters exist which could potentially bring increased insight into mechanisms for cerebral aneurysm behavior; they could also help categorize the severity of such malformations and design effective intravascular treatment techniques. The purpose of this paper is thus to present an overview of such hemodynamic factors that could assist in determining the geometries which present the greatest risks to patients. These parameters include (1) hemodynamic shear stress, (2) pressure and related stresses, (3) impingement force on the aneurysm wall, (4) inflow rate into the aneurysm, and (5) residence time of blood within the aneurysmal sac. In addition, these factors can also be currently estimated in an in vitro setting.

Factors affecting the interaction of tissue factor/factor VII with factor X in a heterogeneous tubular reactor.

A novel reactor recently described for studying phospholipid-dependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further characterized. The reactor is a capillary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s-1 to 1,200 s-1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, Spectrozyme TMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

Further evidence that glycoprotein IIb-IIIa mediates platelet spreading on subendothelium.

In order to explore further the mechanism by which glycoprotein GPIIb-IIIa promotes platelet vessel wall interaction, platelet adhesion to subendothelium was studied in an annular chamber in which subendothelium from rabbit aorta was exposed at a shear rate of 2,600 s-1 to blood from patients with thrombasthenia. Perfusions were conducted for each of 5 exposure times (1, 2, 3, 5 and 10 min), and the percent surface coverage of the vessel segment with platelets in the contact (C) and spread (S) stage was determined. Increased values of platelet contact (C) were obtained in thrombasthenia at all exposure times; this finding is consistent with a defect in platelet spreading, based on a previously described kinetic model of platelet attachment to subendothelium. According to this model of attachment, increased values of platelet contact (C) at a single exposure time may be indicative of either a defect in spreading (S) or initial contact (C), but multiple exposures will result in increased contact only for defects which are related to defective platelet spreading (S). The results obtained over a broad range of exposure times provide more conclusive evidence that GPIIb-IIIa mediates platelet spreading than those previously obtained at single exposure times.

Platelet adhesion, release and aggregation in flowing blood: effects of surface properties and platelet function.

Platelet adhesion to natural and artificial surfaces and adhesion-induced aggregation were investigated in vitro using an annular perfusion chamber. The surfaces were exposed to anticoagulated blood under identical flow conditions (approximately arterial shear rates). The initial attachment of platelets (contact) appeared less surface specific than spreading and release. Fibrillar collagen was the most powerful inducer of platelet degranulation whereas elastin, microfibrils and epon were virtually inactive. Fibrillar collagen caused release also in the absence of spreading. Surface coverage with platelets did not exceed 25% unless spreading occurred. Perfusion with platelet-free plasma or platelet-poor blood did not remove adhering platelets. However, platelets were translocated from mural thrombi to the surface by such perfusion. In addition, platelets which detached from mural thrombi adhered more readily to elastin or microfibrils than platelets from the circulating blood. The initial attachment of platelets to subendothelium was inhibited in von Willebrand's disease, the Bernard-Soulier syndrome and at high concentrations of dipyridamole; spreading was inhibited in storage pool disease of rats, at low temperature (20 degrees C), with EDTA (3 MM) and Prostaglandin E1 (1 muM); and adhesion-induced aggregation was inhibited in thrombasthenia, storage pool disease and after ingestion of sulfinpyrazone or Aspirin. It is concluded that the initial attachment (contact) of platelets, spreading and surface-induced release of platelet constituents are at least partially indendent phenomena, the latter two being highly surface specific. At flow conditions which cause the disappearance of platelet adhesion appears as an irreversible process.

Effect of aspirin and dipyridamole on the interaction of human platelets with sub-endothelium: studies using citrated and native blood.

The effect of aspirin and dipyridamole ingestion on the interaction of platelets with the subendothelium was studied using both citrated blood and directly sampled (native) blood. After obtained control studies, normal human subjects ingested 0.6 g of aspirin, 150 mg of dipyridamole, or a placebo and studies were repeated 1 1/2 hrs later. Subjects continued on placebo, aspirin (0.6 g b.i.d.) or dipyridamole (100 mg q.i.d.) for 6 days and studies were obtained 1 1/2 hrs after the last dose. Blood was circulated through an annular chamber on whose inner core were mounted everted segments of de-endothelialized rabbit aorta. The wall shear rate was 2,600 sec(-1). Surface coverage with adherent platelets and platelet thrombi, as well as several parameters of thrombus dimensions, were evaluated morphometrically. Aspirin ingestion markedly reduced platelet thrombi in citrated blood,--but had a much lesser inhibitory effective in native blood. Platelet adhesion was unaffected in native blood, in contrast to previous findings in which a lower shear rate (800 sec (-1)) was used. Ingestion of dipyridamole did not inhibit platelet adhesion or thrombi in either citrated or native blood. The studies indicated that, with the flow conditions used, aspirin is a relatively weak inhibitor of platelet thrombus formation in directly sampled human blood.

Mechanical factors affecting hemostasis and thrombosis.

Both physical and chemical factors can influence the activity of platelets and coagulation factors responsible for the formation of thrombotic and hemostatic masses in the vicinity of an injured vessel wall. Studies performed in controlled shear devices (viscometers) have indicated that physical factors alone can induce platelet aggregation, even in the absence of exogenous chemical factors. The physical considerations which appear to be important for the local activation of hemostatic/thrombotic mechanisms appear to be related to the magnitude of the shear rate/stress, the duration of the applied physical force and the local geometry. Blood flow alone has multiple influences on platelet and coagulative mechanisms. It has been well established that at physiologically encountered shear conditions, increases in the local shear rate enhance the attachment of platelets to the vessel wall and the growth of platelet aggregates on adherent platelets. In contrast, increases in local shear conditions inhibit the production of fibrin formation on surfaces where tissue factor (TF) is exposed. At levels of shear rate/stress high as compared to normal physiological conditions, but comparable to those observed at the apex of severely stenosed vessels, platelet aggregate formation is dependent on the duration of the exposure time. Considerable advances in our understanding of flow-related mechanisms have evolved from the use of well-defined perfusion chambers employing parallel flow streamlines. However, processes leading to hemostasis and thrombosis generally occur in more complicated flow situations where flow streamlines are not parallel and in which abnormally high, as well as abnormally low, shear rates and shear stress levels may be encountered in close proximity to each other.

Further studies on the presence of functional tissue factor activity on the subendothelium of normal human and rabbit arteries.

Although tissue factor (TF) activity has been observed on the subendothelial surface of rabbit aorta and human umbilical cord, immunofluorescent and in situ hybridization methods have failed repeatedly to demonstrate TF in the intima of human blood vessels. In the present study, TF activity on everted, de-endothelialized arteries was studied by two methods. One utilized a flow system and measured fibrin deposition and fibrinopeptide A formation. The other utilized a newly developed rotating probe system and measured the conversion of factor X to factor Xa in the presence of factor VIIa and Ca+2. The study attempted to control, or assess, the possibility that functional TF could have been exposed on the vessel surface by the procedures used to prepare the arterial segments. By both methods, TF activity was detected on the subendothelium of rabbit aortae and human umbilical arteries, and was unaffected by the length of storage or by inclusion of actinomycin D in the storage buffer. TF activity was also observed in the subendothelium of adult human ileo-colic, internal mammary, and renal arteries, studied by the rotating probe method. The latter may underestimate TF activity, as some of the factor Xa formed appears to bind to the subendothelial surface. TF activity (Xa formation) was detected on the luminal surface (subendothelium) of non-everted arteries, but increased activity was observed after eversion of the vessel. The source of the subendothelial TF, and its presence in normal subendothelium in vivo, requires further study. In addition, if any of the TF activity observed in this study was derived from injured endothelial or myointimal cells during preparation of the everted vessel segments, the techniques described could serve as a useful model for studying TF-induced thrombosis and factor Xa formation on injured blood vessels, and for evaluating the anti-thrombotic properties of TF-inhibitors.

Computer modeling of intracranial saccular and lateral aneurysms for the study of their hemodynamics.

There is strong evidence indicating hemodynamic stress as an underlying cause for saccular intracranial aneurysm growth, thrombosis, and/or rupture. We examined flow fields encountered in models of cerebral aneurysms having a lateral (originating from the side of an artery, not at a branch point) geometric configuration. Shear stress and pressure gradients acting on aneurysm walls under a variety of flow and geometric conditions were evaluated. For this purpose, a two-dimensional finite-element computer model of lateral aneurysms in a steady-flow state was developed. Three idealized aneurysm shapes were studied, half-spherical, spherical, and pear-shaped. The ostium width of the cerebral aneurysm, relative to the radius of the parent artery and the Reynolds number, were also varied. Maximal shear stresses and maximum pressures (for an ostium width of 2 times the radius of the parent artery) were typically found at the downstream site of the ostium, rather than at the dome of the aneurysm. In general, the highest shear stresses and the lowest pressures (at the distal portion of the ostium) were obtained in the spherical aneurysm, whereas the lowest shear stresses and the highest pressures were found in the half-spherical aneurysm. The location of maximal stresses (shear and pressure) at the distal region of the ostium suggests that growth and/or rupture may well proceed from this point. Such findings are in contrast to the commonly held opinion that aneurysm rupture occurs at the dome. Careful pathological investigation will need to be performed to clarify this finding. The results of this preliminary investigation also indicate that the flow field in lateral aneurysms is highly dependent on a number of factors related to flow and geometric parameters. Geometry seems to be a significant mediator of local magnitudes of stress. Thus, the tendency for growth or thrombosis may be influenced by variations in size or shape.