Lesley E. Scudder

Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions.

In an attempt to overcome the limitations and drawbacks of using fresh platelets for transfusion therapy of thrombocytopenic patients, we have performed in vitro experiments on an autologous, semi-artificial alternative to platelet transfusions. Based on our previous studies of the interactions of unactivated and activated platelets with beads coated with peptides of various lengths, all of which contained the arginine-glycine-aspartic acid (RGD) cell recognition sequence, the peptide Ac-CGGRGDF-NH2 was chosen for covalent coupling to erythrocytes. A heterobifunctional crosslinking reagent (N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid) was used to crosslink via the peptides free sulfhydryl group and the erythrocytes surface amino groups. Approximately 0.5-1.5 x 10(6) peptide molecules bound per erythrocyte after 2 h of incubation, and most of the peptides appeared to crosslink to glycophorin A. The resulting cells, termed thromboerythrocytes, interacted selectively with activated platelets to form mixed aggregates. Studies with fluid phase RGD peptides and monoclonal antibodies indicated that the RGD peptides on the thromboerythrocytes interacted with the GPIIb/IIIa receptors on activated platelets. Thromboerythrocytes could also bind to platelets adherent to collagen. There was minimal erythrocyte hemolysis during the formation of thromboerythrocytes and studies of thromboerythrocyte osmotic fragility and cellular deformability showed no significant changes from control erythrocytes. Whereas there is a 20:1 ratio of erythrocytes to platelets in the circulation of normal individuals, the erythrocytes from as little as 50 ml of blood could be transformed into the equivalent of 2 U of platelets by numbers (equivalent to 18 U of platelets by mass), and reinfused into the same individual within several hours. These data encourage us to proceed to in vivo studies to assess the hemostatic efficacy of thromboerythrocytes in thrombocytopenic animals.

Evidence that glycocalicin circulates in normal plasma.

By using a combination of a heterologous antiserum to GPIb/glycocalicin and a radiolabeled monoclonal antibody to GPIb/glycocalicin, we were able to develop a sensitive and specific radioimmunoelectrophoretic assay that can distinguish small amounts of glycocalicin from GPIb. Normal plasmas were found to contain glycocalicin, even in samples treated with protease inhibitors and centrifuged extensively to remove platelets and platelet fragments. Confirmation that the plasma antigen had a relative molecular weight similar or identical to glycocalicin was obtained from studies employing gel chromatography and affinity chromatography. An immunoradiometric assay was developed to quantify plasma glycocalicin, and normal plasma was found to contain approximately 1-3 micrograms/ml. The plasma of a patient with severe thrombocytopenia due to aplastic anemia had less than 12.5% of the normal level of glycocalicin, whereas two patients with thrombocytopenia due to diseases of increased platelet destruction (idiopathic thrombocytopenic purpura and hemolytic-uremic syndrome) had normal levels. Thus, there appears to be ongoing catabolism of platelet GPIb in vivo, and we postulate that the plasma level of glycocalicin reflects a complex function of factors, including platelet count, platelet turnover, and the site of platelet destruction.

Effects of abciximab on the acute pathology of blood vessels after arterial stenting in nonhuman primates

OBJECTIVES This study was designed to assess the effect of abciximab on platelet and leukocyte deposition 60 min after stent insertion in nonhuman primates. BACKGROUND Although it is well established that abciximab improves both short- and long-term clinical outcomes after stent placement, there have been no studies assessing its effect on early platelet and leukocyte deposition. METHODS Cynomolgus monkeys were pretreated with aspirin and either saline or a 0.4 mg/kg bolus of abciximab, and then subjected to angioplasty and Palmaz-Schatz stent placement in the common iliac artery or abdominal aorta. After 60 min, animals were euthanized and the stented artery was evaluated by immunohistochemistry and morphometry. RESULTS Complete occlusion of the stented vessel with a thin fibrin(ogen) meshwork and trapped blood occurred in two saline-treated and two abciximab-treated animals. In the four remaining saline-treated animals, a layer of erythrocytes trapped in a network of fibrin(ogen) was noted close to the vessel wall, and this was covered by a layer of large, irregular platelet thrombi. Leukocytes formed a monolayer on top of the platelets and near stent struts. In the four remaining abciximab-treated animals, the mean erythrocyte area was 65% smaller (p = 0.070), the platelet aggregate area was 89% smaller (p = 0.049) and the luminal area was 59% larger (p = 0.004). A monolayer of leukocytes also formed on top of the platelets and near stent struts. CONCLUSIONS In control stented blood vessels in this study, platelet thrombi formed not at the vessel wall, but on top of an erythrocyte-rich layer, and platelets recruited leukocytes. Abciximab decreased the size of platelet thrombi, but did not prevent leukocyte recruitment.

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

A Shear-restricted Pathway of Platelet Procoagulant Activity Is Regulated by IQGAP1

Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active αIIBβ3 integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm2 (shear rates of 1600 s-1) (p < 0.0001), corresponding to ∼2.5 times the normal shear stress, or ∼40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation (cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca2+]i, but it is specifically coupled to the α-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice

Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.

The organomercurial 4-aminophenylmercuric acetate, independent of matrix metalloproteinases, induces dose-dependent activation/ inhibition of platelet aggregation

Matrix metalloproteinases (MMPs) play an important role in many biological and pathological processes including tissue remodeling, wound healing, inflammation, atherosclerosis, and cancer. Numerous publications have supported the concept that activated MMP-2 enhances agonist-induced platelet aggregation and activated MMP-9 inhibits platelet aggregation. In this study, we demonstrated that the organomercurial compound, 4-aminophenyl mercuric acetate (APMA), which is routinely employed to activate latent MMPs at a concentration of 1000 microM, induces platelet aggregation at low concentration (5 microM) and inhibits agonist-induced platelet aggregation at concentrations >or= 50 microM. Activated MMP-2, MMP-1, and MMP-9, following removal of APMA by ultrafiltration through an anisotropic membrane, exert no independent effect on platelet aggregation. Acetylsalicylic acid and BAPTA inhibited APMA-induced platelet aggregation indicating that the APMA mediated pathway of platelet activation is dependent upon thromboxane and calcium signaling. Zinc chelation with 1,10-phenanthroline, which inhibits zinc-dependent proteins including metalloproteinases, also abrogated platelet functional responses to APMA. Additional studies will be required to clarify the mechanism of the biphasic effect of APMA on platelet aggregation.