Arginine-glycine-aspartic acid- and fibrinogen gamma-chain carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wall shear rates. An effect dissociable from interference with adhesive protein binding.

Arg-Gly-Asp (RGD)- and fibrinogen gamma-chain carboxyterminal (GQQHHLGGAKQAGDV) peptides inhibit fibrinogen, fibronectin (Fn), vitronectin, and von Willebrand factor (vWF) binding to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). GP IIb-IIIa, vWF, and Fn are essential for normal platelet adherence to subendothelium. We added peptides to normal citrated whole blood before perfusion over human umbilical artery subendothelium and evaluated platelet adherence morphometrically at high (2,600 s-1) and low (800 s-1) wall shear rates. We also examined the effects of the peptides on platelet adhesion to collagen in a static system. At the high wall shear rate, RGDS and GQQHHLGGAKQAGDV caused dose-dependent reduction in the surface coverage with spread and adherent platelets. Amino acid transposition and conservative substitutions of RGD peptides and the AGDV peptide significantly inhibited platelet adherence at 2,600 s-1. By contrast, the modified RGD peptides and AGDV do not affect adhesive protein binding to platelets. None of the native or modified RGD- or fibrinogen gamma-chain peptides significantly inhibited either platelet adherence to subendothelium at 800 s-1 or platelet adhesion to collagen. Our findings demonstrate that peptides that interfere with adhesive protein binding to GP IIb-IIIa inhibit platelet adherence to vascular subendothelium with flowing blood only at high wall shear rates. Platelet adherence to subendothelium at high wall shear rates appears to be mediated by different recognition specificities from those required for fluid-phase adhesive protein binding or static platelet adhesion.     

Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.     

A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components

A flat perfusion chamber was developed to study the interaction of blood platelets in flowing blood with cultured human vessel wall cells, their connective tissue matrix, and isolated connective tissue components at defined shear rate conditions. A cover slip covered with endothelial cells or extracellular matrix components was introduced into the chamber. Laser-Doppler velocimetry showed a symmetrical flow profile at flow rates between 50 and 150 ml/min (wall shear rate 300 to 1100 sec-1). Platelet deposition was estimated by using blood platelets labeled with indium-111 or by a morphometric method. Blood platelets did not adhere to endothelial cells at wall shear rates of 765 sec-1 and the endothelial cells remained attached for at least 10 min of perfusion. In preconfluent cultures of endothelial cells, blood platelets adhered to extracellular material in areas between the cells. Removal of endothelial cells by treatment with 0.5% Triton X-100 induced increased platelet adherence with a preference for certain, as yet unidentified, fibrillar structures of the extracellular matrix. Platelet adherence to equine collagen was also studied after coating the cover slips by spraying of small collagen droplets followed by air drying. Platelet adherence and the subsequent platelet aggregate formation occurred predominantly along visible collagen fibers. These studies showed that this perfusion chamber has a laminar and symmetrical flow allowing qualitative and quantitative investigation of platelet interaction with endothelial cells, their extracellular matrix, and pure connective tissue components. A variety of wall shear rates and exposure times can be applied at controlled conditions without removing cells or extracellular material.     

Increased platelet deposition on atherosclerotic coronary arteries.

A ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-microns cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. The central lipid core of an advanced plaque was not reactive towards platelets. The results indicate that the atherosclerotic plaque by itself is more thrombogenic than the normal vessel wall. To study the cause of the increased thrombus formation on the atherosclerotic plaque, perfusion studies were combined with immunohistochemical studies. Immunohistochemical studies of adhesive proteins showed enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. Laminin and collagen type IV were not enriched. von Willebrand Factor (vWF) was not present in the plaque. The pattern of increased platelet deposition in serial cross sections corresponded best with areas in which collagen types I and III were enriched, but there were also areas in the plaque where both collagens were enriched but no increased reactivity was seen. Inhibition of platelet adhesion with a large range of antibodies or specific inhibitors showed that vWF from plasma and collagen types I and/or III in the plaque were involved. Fibronectin from plasma and fibronectin, fibrinogen, laminin, and thrombospondin in the vessel wall had no effect on platelet adhesion. We conclude that the increased thrombogenicity of atherosclerotic lesions is due to changes in quantity and nature of collagen types I and/or III.     

Thrombospondin-platelet interactions. Role of divalent cations, wall shear rate, and platelet membrane glycoproteins.

The role of thrombospondin, a multifunctional matrix glycoprotein, in platelet adhesion is controversial: both adhesive and antiadhesive properties have been attributed to this molecule. Because shear flow has a significant influence on platelet adhesion, we have assessed thrombospondin-platelet interactions both under static and flow conditions. The capacity of thrombospondin to support platelet adhesion depended upon its conformation. In a Ca(2+)-depleted conformation, such as in citrated plasma, thrombospondin was nonadhesive or antiadhesive as it inhibited platelet adhesion to fibrinogen, fibronectin, laminin, and von Willebrand factor by 30-70%. In a Ca(2+)-replete conformation, however, thrombospondin effectively supported platelet adhesion. Shear rate influenced this adhesion; percent surface coverage on thrombospondin increased from 5.4 +/- 0.3 at 0 s-1 to 41.5 +/- 6.7 at 1,600 s-1. In contrast to the extensive platelet spreading observed on fibronectin at all shear rates, platelet spreading on thrombospondin occurred only sporadically and at high shear rates. GPIa-IIa, GPIIb-IIIa, GPIV, and the vitronectin receptor, which are all proposed platelet receptors for thrombospondin, were not solely responsible for platelet adhesion to thrombospondin. These results suggest that thrombospondin may play a dual role in adhesive processes in vivo: (a) it may function in conjunction with other adhesive proteins to maintain optimal platelet adhesion at various shear rates; and (b) it may serve as a modulator of cellular adhesive functions under specific microenvironmental conditions.     

alphaIIbbeta3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium.

Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antialpha(IIb)beta(3) antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that alpha(IIb)beta(3) antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s(-1), largely VWF-independent. When perfused over a human umbilical vein endothelial cell-derived extracellular matrix, single alpha(IIb)beta(3)-antagonized platelets primarily adhered to matrix-bound VWF when perfused at 2700 s(-1), but at 1300 s(-1) they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de-endothelialized rabbit aorta at a wall shear rate of 1100 s(-1), alpha(IIb)beta(3) antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected alpha(IIb)beta(3)-antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, alpha(IIb)beta(3) antagonism inhibited platelet-rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than alpha(IIb)beta(3) antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.     

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.     

Platelet adhesion to multimerin 1 in vitro: influences of platelet membrane receptors, von Willebrand factor and shear

Summary.  Background : Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein, stored in platelets and endothelium, that when released, binds to activated platelets, endothelial cells and the extracellular matrix. Objectives : The goals of our study were to determine if (i) MMRN1 supports adhesion of resting and/or activated platelets under conditions of blood flow, and (ii) if MMRN1 enhances platelet adhesion to types I and III collagen. Patients / methods : Platelet adhesion was evaluated using protein-coated microcapillaries, with or without added adhesive proteins and receptor antibodies. Platelets from healthy controls, Glanzmann thrombasthenia (GT) and severe von Willebrand factor (VWF)-deficient donors were tested. Results : MMRN1 supported the adhesion of activated, but not resting, washed platelets over a wide range of shear rates. At low shear (150 s⁻¹), this adhesion was supported by integrins αvβ3 and glycoprotein (GP) Ibα but it did not require integrins αIIbβ3 or VWF. At high shear (1500 s⁻¹), adhesion to MMRN1 was supported by β3 integrin-independent mechanisms, involving GPIbα and VWF, that did not require platelet activation when VWF was perfused over MMRN1 prior to platelets. MMRN1 bound to types I and III collagen, independent of VWF, however, its enhancing effects on platelet adhesion to collagen at high shear were VWF dependent. Conclusions : MMRN1 supports platelet adhesion by VWF-dependent and -independent mechanisms that vary by flow rate. Additionally, MMRN1 binds to, and enhances, platelet adhesion to collagen. These findings suggest that MMRN1 could function as an adhesive ligand that promotes platelet adhesion at sites of vascular injury.     

Alcohol and polyphenolic grape extract inhibit platelet adhesion in flowing blood

BACKGROUND: Moderate and prolonged alcohol consumption has been associated with decreased cardiovascular morbidity and mortality. Inhibition of platelet function in suspension attributes to these effects. Whether alcohol, red wine, or polyphenolic grape extracts (PGE) inhibit platelet adhesion is not known. We investigated platelet adhesion to fibrinogen and collagen in whole blood under standardised flow. MATERIALS AND METHODS: Before perfusion was started, citrated whole blood from 95 volunteers was preincubated for five min with different alcohol concentrations, unfractioned red wine and PGE. Then, blood was perfused in a single-passage flow chamber over coverslips coated with human fibrinogen or collagen type III at shear rates of 300 s(-1) and 1600 s(-1). RESULTS: Alcohol inhibited platelet adhesion to human fibrinogen at high shear rate (concentrations > or = 0.15 per thousand) and low shear rate (only at a concentration of 4.8 per thousand), whereas red wine (concentrations > or = 0.15 per thousand) inhibited platelet adhesion to human fibrinogen at both shear rates. In contrast, PGE (concentrations > or = 0.0225 g L(-1)) inhibited platelet adhesion to human fibrinogen only at low shear rate. None of these incubations affected adhesion to collagen. CONCLUSIONS: Alcohol, red wine and PGE inhibit adhesion to fibrinogen but not to collagen. This inhibition might contribute to the cardioprotective effects of moderate alcohol consumption.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.     

von Willebrand factor and the blood vessel wall

The role of von Willebrand factor (vWF) in mediating platelet adhesion has been established with the use of in vitro perfusion systems. vWF binds to the subendothelium, changes in conformation, and is then able to interact with glycoprotein Ib. vWF deposited in the subendothelium is responsible for up to 40% of normal adhesion. The action of vWF is seen at high shear rates. It also acts at low shear rates, but other factors can assume its role. Binding of vWF to the subendothelium is not through the established collagen binding sites but involves a new domain on the vWF molecule. Collagen VI has been implicated as the vessel wall component involved. Deposition of vWF in the subendothelium is determined by growth conditions and activation state of the endothelial cell. Subendothelial vWF in von Willebrand's disease may support platelet adhesion in situations in which plasma vWF of the same patient does not.     

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.     

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10-fold more potent than acid-soluble collagen in inducing platelet aggregation and found that acid-soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid-soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid-soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme-linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody-coated beads and to agglutinate GPIb-IX-V complex-expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen-induced platelet aggregation, aggregation induced by acid-soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIbalpha antibody that inhibits the GPIb-VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagen-dependent platelet functions and further emphasizes the close synergistic roles of the GPIb-IX-V complex and the collagen receptors GPVI and alpha2beta1 in supporting platelet adhesion.     

Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear‐resistance of platelet aggregates

Summary. Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1–10 mL of blood and are poorly suited for murine whole blood experiments. Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 μL of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid‐soluble collagen in 100 μm × 100 μm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti‐mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin α₂ subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a ‘shear step‐up’ to 8000 s^?1. Results: Wild‐type murine platelets adhered and aggregated on collagen in a biphasic shear‐dependent manner with increased deposition from 100 to 400 s^?1, but decreased deposition at 1000 s^?1. Adhesion to patterned collagen was severely diminished for platelets lacking a functional α₂β₁ integrin. Those integrin α₂‐deficient platelets that did adhere were removed from the surface when challenged to shear step‐up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step‐up. Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin‐dependent pathways such as fibrin formation.     

von Willebrand factor-mediated platelet adhesion to collagen involves platelet membrane glycoprotein IIb-IIIa as well as glycoprotein Ib

Monoclonal antibodies (MAbs) directed toward distinct functional domains of human von Willebrand factor (vWF) were used to probe the involvement of platelet membrane receptors glycoprotein Ib (GPIb) and glycoprotein IIb-IIIa (GPIIb-IIIa) in vWF-mediated platelet adhesion to collagen in flowing blood. Among nine MAbs to vWF, MAb H9 inhibits binding of vWF to GPIb, MAb 9 blocks binding of vWF to GPIIb-IIIa, and MAbs B200 through B204 inhibit binding of vWF to collagen. Collagen-coated cover slips were exposed to human citrated blood at shear rates varying from 200 to 2600 sec-1 in parallel-plate perfusion chambers. Blood was reconstituted with washed radiolabeled platelets, erythrocytes, and citrated autologous plasma previously incubated with MAb IgG or F(ab')2. Platelet-collagen interactions were estimated by radioactive counting and by quantitative morphometry. Inhibition of 70% of platelet adhesion was observed at a concentration of 20 micrograms/ml MAb 9, similar to that observed with MAbs H9 or B202. The effect of each MAb was dose dependent, and their inhibitory effect on platelet adhesion was also shear rate dependent. Virtually 100% inhibition was observed at 2600 sec-1 shear rate when a mixture of the MAbs (H9 + 9 + B202) was added to reconstituted blood. Thus, blocking three functional domains of vWF virtually abolishes platelet-collagen adhesion at high shear rates, indicating that normal adhesion to collagen is mediated through binding of vWF to collagen and to both platelet membrane GPIb and GPIIb-IIIa.     

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.     

Gray platelet syndrome: alpha-granule deficiency. Its influence on platelet function

The ultrastructure, cytochemistry, biochemistry, and functions of platelets from two patients with the familial gray platelet syndrome are described. Ultrastructural studies showed a lack of alpha-granules in the megakaryocytes in the vicinity of a marrow myelofibrosis and/or in platelets. A normal number of mitochondria and a slight increase of dense bodies was confirmed by labeling the whole patient with mepacrine. Platelet peroxidase (in the dense tubular system) and catalase-positive granules (revealed by the cytochemistry) were present. Platelets from both patients had severe deficiency of beta TG either after tritonization (less than 4% of normal) or thrombin treatment (less than 15% of normal), and the plasma beta TG levels were slightly increased. Functional studies of these platelets showed an uptake of [14C]5HT inhibited by reserpine similar to that in the control platelets. Thromboxane formation was within normal limits in the presence of arachidonic acid, ADP, collagen, or ionophore A23187, indicating that (1) the cyclooxygenase/thromboxane synthetase systems were not altered and (2) the phospholipase activities were not impaired. Although the platelet adhesion to prepolymerized fibrillar type III collagen and the ADP-, arachidonic acid-, and ionophore A23187-mediated aggregations were apparently normal, in every case the release of [14C]5HT was either at the low side of the normal range or decreased. This abnormality was increased when collagen or thrombin was added to either PRP or washed platelets from both patients, where at the same time, the aggregation and the release were markedly reduced. The results suggest that alpha-granules or their content has an influence on not only the platelet aggregation but also the dense bodies involved in the [14C]5HT release reaction.     

Haemostasis, inflammation and renal function following exercise in patients with intermittent claudication on statin and aspirin therapy

Background

Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system.

Methods

Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s^-1 to 1500 s^-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated.

Results

Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s^-1 and 50 s^-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 ± 3.3%) and high (117 ± 4.1%) antithrombin activity (1786 ± 516 U vs. 823 ± 331 U, p < 0.05) at low flow rate (13 s^-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4 p = 0.008, respectively).

Conclusion

It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.     

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen

Summary.  Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb–IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb–IIIa and P-selectin than those on VWF. Activation of GpIIb–IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca²⁺ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb–IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.     

Monocytes downregulate the early stage of collagen-induced platelet activation by a PECAM-1-dependent mechanism

Summary.  Background:  Blood vessel damage results in exposure of the subendothelial matrix, to which platelets adhere. Monocytes are recruited and activated at the site of injury. Objectives:  Here we studied the effect of monocytes on platelet activation induced by exposure to fibrillar collagen. Methods:  Washed platelets and isolated monocytes (100/1) were coincubated with type I collagen in static adhesion conditions or in suspension. Platelet activation was assessed by measuring RANTES production and α-granule secretion. Platelet adherence on immobilized collagen was analyzed by fluorescence confocal microscopy. Cell–cell contacts were prevented by incubating platelets and monocytes in transwell coculture dishes. Experiments were also performed in the presence of soluble recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1) or of antibodies to PECAM-1. Results:  Unexpectedly, unstimulated monocytes limited the initial phase of platelet activation by fibrillar collagen. In adhesion conditions, monocytes reduced the secretion by platelets of the inflammatory chemokine RANTES and of β-thromboglobulin and the formation of platelet aggregates. The inhibitory effect of monocytes on platelet activation required direct cell–cell contacts between platelets and monocytes. Monocytes also inhibited collagen-induced platelet activation in suspension conditions as assessed by the reduction of P-selectin exposure and RANTES secretion. A recovery of platelet responses was observed in the presence of soluble PECAM-1 and of PECAM-1.3 Fab, indicating that PECAM-1 is involved in monocyte-triggered downregulation of platelet reactivity. Conclusions:  Our data provide the first evidence that unstimulated monocytes limit the initial phase of platelet activation by collagen via a mechanism that is, at least in part, PECAM-1-dependent.