The effect of fibrinogen sialic acid residues on ex vivo platelet deposition on biomaterials

The effect of fibrinogen sialic acid residues on platelet deposition onto polymer surfaces was examined using a canine ex vivo shunt model. To test the hypothesis that desialylated fibrinogen may enhance platelet deposition when precoated on biomaterials, canine fibrinogen was desialylated and precoated on polyvinyl chloride (PVC) shunts. When protein-coated PVC shunts were exposed to flowing whole blood, both the native and the desialylated fibrinogen elicited the same profile of platelet deposition. This study indicates that platelet deposition and thrombus formation on biomaterial surfaces is not mediated by a mechanism which involves the sialic acid residues of fibrinogen.     

Thrombin stimulated platelet accumulation on protein coated glass capillaries: role of adhesive platelet alpha-granule proteins

The generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22 degrees C from solutions of thrombin 100 NIH units (33 micrograms)/ml gave surface concentrations in the range 0.019-0.101 micrograms/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37 degrees C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s-1 or 2,000 s-1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet alpha-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s-1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by anti-thrombospondin antibodies.     

Measurement of transient thrombus deposition on polymeric materials

The method developed to measure transient thrombus deposition from arterial blood on polymeric surfaces involves infusion of small amounts of ¹²⁵I-labeled fibrinogen and ⁵¹Cr-labeled platelets into the circulation of anesthetized mongrel dogs. Radioactivity adsorbed to the walls of femoral A-V shunts was counted to measure fibrin and platelet deposition with time following initial blood-polymer contact. Shunt segments were obtained at each datum for scanning electron microscopy of adhered thrombus material. Plasticized polyvinyl chloride (PVC), recognized as having poor blood compatibility, elicited rapid platelet and fibrin activation resulting in a transient maximum of thrombus deposition after 10 to 15 minutes of blood contact. Adherent thrombi subsequently left the surface. Contact of blood with segmented polyetherurethane (BIOMER) resulted in minimal platelet and fibrin deposition. Morphological analysis by electron microscopy confirmed these observations. This method can be used to determine the thrombogenic and embolic potential of polymeric surfaces and may be used to elucidate the mechanisms of artificial surface induced thrombosis.     

Surface-dependent expression in the platelet GPIb binding domain within human von Willebrand factor studied by atomic force microscopy

Adsorption of plasma proteins such as von Willebrand factor (vWF) on thrombogenic surfaces can induce conformational changes in tertiary structure so that the prothrombotic functional epitopes are exposed for interactions with platelets, resulting in platelet adhesion and thrombus formation. Thus, understanding platelet binding following changes in the structure of vWF is critical in understanding the mechanisms of thrombogenesis. The present study examined the accessibility of platelet binding epitopes within vWF adsorbed on two different thrombogenic surfaces, a hydrophobic synthetic surface and collagen VI coated substrates, under physiological buffer conditions using atomic force microscopy (AFM) in combination with immunogold labeling. Our results demonstrated that the glycoprotein Ib (GPIb) binding domain in vWF undergoes changes when adsorbed on collagen VI compared to vWF on a hydrophobic synthetic surface. This study provides a basis for a novel approach to understand the molecular mechanisms of surface-induced thrombosis by directly examining the structure–function relationships of plasma proteins involved in the thrombus formation.     

Glycocalicin binding to von Willebrand factor adsorbed onto collagen-coated or polystyrene surfaces

In order to analyze the interaction of platelets with von Willebrand factor (vWF) and collagen, we studied the binding of glycocalicin (GC) and formalin-fixed platelets to vWF adsorbed onto uncoated or collagen-coated polystyrene surfaces. These studies show that three-fold more vWF binds to collagen-coated polystyrene than to polystyrene coated with fibrin monomer or fibrinogen. At saturation, 37 +/- 2.9 ng vWF bound to the collagen-coated wells, compared to 12.8 +/- 5.4 ng, and 10.9 +/- 2.7 ng of vWF bound to wells coated with fibrin monomer and fibrinogen, respectively. GC also bound significantly more to collagen-coated wells than to wells coated with fibrinogen, and this binding was increased approximately two-fold (from 7 +/- 0.65 ng to 14 +/- 1.1 ng) in the presence of vWF adsorbed to the collagen-coated surface. Only 2 ng of GC was bound to 3000 ng of vWF when the latter was adsorbed directly onto a polystyrene surface. In contrast, GC binding to vWF adsorbed onto a collagen-coated surface was enhanced 600-fold with 7.0 ng of GC bound to 18 ng of immobilized vWF. Formalin-fixed platelets showed little binding to vWF adsorbed onto the microtiter wells. At saturation, 7 x 10(4) platelets bound to 3000 ng of vWF; a 6-fold increase in platelet binding was observed using collagen-coated wells and this binding was increased even further in the presence of vWF, resulting in 250-fold increase in platelet binding to vWF when the latter was adsorbed onto a collagen surface. These studies suggest that (1) GC is involved in platelet binding to collagen and this binding is increased by vWF; (2) GC binding to vWF is enhanced by the collagen-coated surface; (3) the adsorption of vWF onto a collagen surface may induce conformational changes in vWF that promote its interaction with GC or glycoprotein Ib.     

The use of monoclonal antibodies to human platelet membrane glycoprotein IIb-IIIa to quantitate platelet deposition on artificial surfaces

A new technique is described to quantitate platelet deposition in vitro on artificial surfaces, based on a surface phase radioimmunoassay using the monoclonal antibody 6C9, directed specifically against the membrane glycoprotein complex IIb-IIIa of human platelets. Results correlate in linear fashion with those obtained using 111Indium labeled platelets. The method offers particular advantages for the measurement of platelet deposition in whole blood, since platelet separation, washing and labeling procedures are eliminated, together with the ensuing possible selection of platelet populations. In vitro perfusion is performed in glass capillaries of precisely defined diameter (0.80 or 0.56 mm i.d.). Blood flow is laminar and accurately controlled over wall shear rates ranging from venous to capillary (50-4,000 s-1). Using glass capillaries precoated with purified human albumin or fibrinogen or bovine collagen, platelet deposition from suspensions of washed human platelets in Tyrode's-albumin buffer in the presence of a 40% hematocrit is (platelets/mm2): 11,000 (albumin), 78,000 (fibrinogen) and 306,000 (collagen) after 5 min perfusion at 2,000 s-1. In heparin, citrate or hirudin anticoagulated whole blood, surfaces are passivated, probably by albumin adsorption from plasma (platelets/mm2): 400 (albumin), 3,600 (fibrinogen) and 48,000 (collagen) after 5 min perfusion in the presence of 13 mM citrate.     

Absence of fibrinogen in afibrinogenemia results in large but loosely packed thrombi under flow conditions

We studied the role of fibrinogen in platelet thrombus formation under flow on adhesive proteins using afibrinogenemic blood (LMWH anticoagulated) in a perfusion system. Perfusions with afibrinogenemic blood showed strong increased surface coverage and thrombus volume that normalized upon addition of fibrinogen. Similar studies using citrate anticoagulated blood showed that this was due to fibrinogen and not fibrin. Morphological analysis showed that afibrinogenemic thrombi were loosely packed and consisted mainly of dendritic platelets that contacted one another through filopodia. However, in the presence of fibrinogen, platelets formed lamellipodia and spread out on top of one another. Studies with radiolabeled platelets showed similar numbers of platelets in both conditions demonstrating that the difference is one of packing and the larger size is due to absence of lamellipodia formation and spreading. The found increased thrombus size and loosely packed platelets might help to understand thrombotic complications sometimes seen in afibrinogenemia patients.     

A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic

The thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 microg/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.     

Growth and stability of thrombi in flowing citrated blood: assessment of platelet-surface interactions with computer-assisted morphometry

The differential quantitation of platelet deposition in perfusion studies is a major problem. We report on methods to prepare semithin sections of platelet deposits on collagen coated on glass and plastic cover slips, to study growth and stability of thrombi in three dimensions, and the development of a computer-assisted differential quantitation of platelet-collagen interactions. The interactions were quantified as percentage of the surface covered with platelets (platelet adhesion), thrombus height, thrombus density and thrombus area per unit sectional length, respectively. Cover slips coated with fibrillar equine collagen in parallel-plate perfusion chambers were exposed to flowing citrated blood at shear rates ranging from 200 to 2,600 s-1. Thrombi, partially enmeshed in the collagen meshwork, prevailed on the surface at all shear rates. Maximal platelet adhesion and thrombus density were seen at greater than 5 micrograms/cm2 collagen, while thrombus area and height were maximal at greater than 10 micrograms/cm2. The volume of the thrombi appeared correlated to the number of deposited platelets (r = 0.92). En face preparations showed deposits of platelet islands which grew in diameter with time, particularly in the direction of the blood flow, becoming progressively confluent. Sections cut parallel to the direction of the blood stream indicated that this growth pattern was at least partially caused by thrombi bent in the direction of the blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinogen adsorption and platelet adhesion to metal and carbon coatings

In order to study the haemocompatibility of metal and carbon coatings, fibrinogen adsorption and platelet adhesion to various coatings have been investigated. Two metallic coatings--titanium and zirconium, and two carbon coatings - isotropic diamond-like and isotropic graphite-like coatings, were prepared by plasma vapour deposition onto stainless steel substrate. It has been shown that the adsorption of fibrinogen to metal and carbon coatings and its post-adsorptive transition are dependent on both the material properties and the fibrinogen environment. The adsorption of fibrinogen from human plasma on titanium and zirconium coatings is similar to that on uncoated stainless steel surface. Both carbon coatings adsorb much greater amount of fibrinogen from plasma, and fibrinogen retention by carbon surfaces is also greater than by metal surfaces. Increased numbers of adhered platelets have been found on both carbon coatings in comparison to the metal materials, although this does not correlate with the amount of adsorbed fibrinogen.     

Fibrinogen, fibrin and crosslinking in aging arterial thrombi

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous (111)In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37 degrees C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from (111)In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bbeta- and gamma-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of gamma-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.     

Electric injury model of murine arterial thrombosis

BACKGROUND AND PURPOSE: Murine models of arterial thrombosis have gained utility with applications in genetically manipulated mice. Implementation of current models requires specialized equipment and provides limited outcome measures. A new murine model of continuously monitored arterial thrombosis was created. METHODS: An electric injury was delivered to the exterior surface of the common carotid artery using the flat end of a 140-mum steel microsurgery needle connected to the anode of a 3-V battery source. Direct current was delivered for 30 s. The developing thrombus was apparent as a white, platelet-dominated region at the site of injury. This region was continuously monitored and recorded by videotape for 30 min. Subsequently, the thrombus area was measured directly on the TV monitor, generating a time course for thrombogenesis. In a further evaluation of the model, three pharmacologically treated groups of mice were evaluated, with drug infusion immediately before thrombus induction: (1) saline (control), (2) heparin (60 units/kg), and (3) GR144053, a GPIIb/IIIa-specific antagonist (10 mg/kg). RESULTS: The basic model showed consistent thrombus development by 7-9 min, occasionally forming an occlusive thrombus. Most of the thrombi underwent one or more cycles of embolization and thrombus regrowth. In the experimental series, the heparin-treated group had a significantly decreased thrombus area versus controls (p<0.0001); the GR144053-treated mice had no apparent thrombus, supporting a dominant role of platelet aggregation in arterial thrombogenesis. CONCLUSION: This new model is simple to do, uses readily available instrumentation, and provides a continuously recorded quantifiable measure of thrombogenesis.     

Studies of suloctidil in experimental thrombosis in baboons

Suloctidil has been evaluated in the baboon for its antithrombotic efficacy using models of both acute and chronic arterial thrombogenesis. Acute thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous shunts. 111In-platelet deposition was measured by scintillation camera imaging for one hour. The results after oral administration of suloctidil (100 mg/kg/d in two divided doses) were not different from control studies. Moreover, concurrent heparin anticoagulation did not affect 111In-platelet deposition compared with control data. In contrast, ticlopidine (20 mg/kg/d) significantly decreased platelet deposition that was reduced further by the addition of heparin. Chronic arterial-thromboembolism was initiated by segments of polyurethane (Biomer) cannula introduced into chronic arteriovenous shunts. Thrombus formation by the polyurethane cannula was measured as 111In-platelet turnover (corrected for removal of senescent platelets). Cannula platelet consumption was unaffected by suloctidil (20 mg/kg/d given in two divided doses for two days preceding and throughout the period of platelet survival measurement). In contrast, dipyridamole (10 mg/kg/d) and sulfinpyrazone (100 mg/kg/d) completely interrupted cannula platelet consumption. We conclude that suloctidil probably has little or no effect on platelet-dependent thrombus formation.     

Vascular injury and thrombosis: a scanning electron microscopic study

Injury to the blood vessel wall and atherosclerosis enhance the possibility of thrombus depostion. A knowledge of the changes in vascular morphology and the extent of thrombus deposition produced by injury is essential in elucidating the mechanism of this reaction. In the present work, scanning electron microscopic technique was used to examine the canine blood vessel wall at or near a site of injury caused by vascular clamping. Proximal to the clamping, the endothelial folds in normal vessels are clearly visible. The intima is completely severed at the site of clamping. At a distance of 1mm distal to the injury, the vascular wall is completely covered with a layer of thrombi, composed mainly of platelets, fibrin and trapped erythrocytes. Thus, injury to the blood vessel wall destroys the endothelial structure and triggers thrombus deposition.     

Expression of the platelet procoagulant activity in vivo in thrombus formation in an extracorporeal shunt in the rat

The interaction of platelets and the coagulation system has been investigated $\text{in vivo}$ on a cotton thread implanted in an arteriovenous shunt in the rat. Depletion of the circulating platelet count with anti-platelet antibody reduces both thrombus deposition and ¹²⁵I-labelled fibrinogen incorporation demonstrating the platelet procoagulant activity $\text{in vivo}$. Inhibition of platelet behaviour with either anagrelide or prostacyclin decreases thrombus deposition by a greater extent than that expected from the platelet content of the thrombus. Treatment with warfarin also dramatically decreases both formation and ⁵¹Cr-labelled platelet deposition demonstrating the important role of the coagulation system in platelet aggregation in this model. Treatment with Arvin or with heparin also reduced the thrombus weight. It is concluded that this is a model of bidirectional platelet interaction with the coagulation system and may have similarities with thrombosis which occurs in extracorporeal shunts in man.     

An in vitro evaluation of the effect of laser irradiation on the thrombogenicity of thrombus.

The effect of laser irradiation on the thrombogenicity of thrombus was evaluated by treating thrombi, formed in-vitro from canine blood, with two different doses of cw Nd:YAG laser energy at 1064 nm. The thrombi were then incubated with whole blood, and the plasma levels of fibrinogen and thrombin-antithrombin III-complexes were measured. A statistically significant decrease (p < 0.05) in the thrombogenicity was indicated by a reduction in both fibrinogen consumption and levels of thrombin-antithrombin III-complexes in the high dose group (600 joules, 100 degrees C peak temperature) in comparison to the low dose group (300 joules, 70 degrees C peak temperature) and the untreated thrombi. These findings suggest that laser irradiation of thrombus at an appropriate dose may substantially reduce its thrombogenicity and ability to modulate hemostasis.     

Platelet physiology and thrombosis

Glycoprotein (GP) Ib of the GPIb-IX–V complex and GPVI bind von Willebrand factor (vWF) and collagen, respectively, and are critical for the initial interaction of circulating platelets with the injured vessel wall under high shear conditions. These interactions act together to facilitate stable thrombus formation in vivo. Ligand binding to GPIb-IX–V of the leucine-rich repeat family or GPVI of the immunoglobulin superfamily initiates platelet activation, and inside–out activation of the platelet integrin, IIbβ3, that binds vWF or fibrinogen and mediates platelet aggregation. The binding site for GPIb on vWF resides in the conserved A1 domain, encompassing the disulfide bond at Cys509–Cys695. This domain may be activated to bind platelet GPIb under shear stress by anchoring of the downstream A3 domain to collagen and conformational distortion of the intervening A2 domain. The N-terminal, 282 residues, of GPIb contains the binding site for vWF-A1, as well as the conserved A-type domain of the leukocyte integrin Mβ2 (M I domain) and P-selectin expressed on activated platelets or endothelial cells. Endothelial P-selectin also supports surface expression of vWF multimers, enabling platelet vessel wall interaction by at least two mechanisms. Recent evidence suggests GPVI that binds collagen, and GPIb-IX–V that binds collagen-bound vWF are physically associated on the platelet surface. This review will focus on the structure–function of primary platelet adhesion receptors, GPIb-IX–V and GPVI, and how they act together to regulate platelet thrombus formation in pathophysiology.     

The subendothelium of the HMEC-1 cell line supports thrombus formation in the absence of von Willebrand factor and collagen types I, III and VI

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1's ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1's ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2,600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC's ECM. Interestingly, large thrombi were formed at 2,600/s when HMEC-1's ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1's ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.     

Platelet compatibility of an artificial surface modified with functionally active heparin.

Platelet compatibility after coating an artificial material with functionally active heparin was investigated. Blood was circulated in uncoated or heparin coated PVC tubing. In one hour platelet counts decreased from 155 (113-184)x10(9)/l to 124 (100-148)x10(9)/l with uncoated compared to 164 (132-192)x10(9)/l with heparin coated tubing (intergroup p = 0.02). Beta-thromboglobulin increased from 116 (80-148) microg/l to 1039 (757-1298) microg/l with uncoated and to 352 (229-638) microg/l with heparin coated tubing (intergroup p = 0.005). Platelet counts and beta-thromboglobulin correlated closely with complement activation. Solid-phase enzyme immunoassay demonstrated substantial deposition of CD42a/GPIbIX and CD61/GPIIIa on uncoated, but not on heparin coated tubing (intergroup p<0.0005). Scanning electron microscopy demonstrated activated platelets and aggregates on uncoated in contrast to heparin coated tubing, where scattered, unactivated platelets were found. Changes in P-selectin and microparticles were minor. In conclusion, this heparin surface substantially improved platelet compatibility. Markers of choice for in vitro evaluation were platelet counts, beta-thromboglobulin and platelet deposition.     

Evaluation of antithrombotic properties of suloctidil in comparison with aspirin and dipyridamole

The antithrombotic effect of suloctidil, a new antispasmotic with platelet anti-aggregating properties was evaluated in two rabbit models. In the first, thrombi were formed in polyethylene catheters placed in the lumens of the carotid arteries. Quantitation of the thrombi was achieved with radio-labelled fibrinogen. The antithrombotic effect of suloctidil was significantly greater than placebo or aspirin and insignificantly greater than intravenously administered dipyridamole. In the second model, two appropriately spaced doses of endotoxin were given. Suloctidil did not inhibit the initial endotoxin induced consumption of platelets or WBC, but did inhibit the second fall in platelets believed related to thrombin elaboration. An accompanying slight inhibition of fibrin deposition and fibrinogen consumption was found.