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Publications and source records attributed to B Shenkman.
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Exposure of whole blood (WB) to subendothelial extracellular matrix (ECM) under shear stress in the cone and plate(let) analyser (CPA) results in platelet adhesion, followed by release reaction and aggregation of circulating platelets on the adherent platelets. The properties of circulating non-adhered platelets in the CPA was studied by exposure of WB to ECM at a high shear rate (1300/s) for 2 min (1st run), followed by transfer of the suspension to a new ECM-coated well for a second run (2nd run) under similar conditions. The results of the 2nd run demonstrated transient adhesion refractoriness associated with platelet microaggregate formation in the suspension. The adhesion refractoriness was dependent on platelet activation during the 1st run and was prevented by addition of apyrase (ADP scavenger) or ADP receptor inhibitor, suggesting a role for ADP in mediating this response. Furthermore, exposure of WB samples to suboptimal concentrations of ADP (0.4-1 micromol/l) or a thrombin receptor activating peptide (TRAP) (5 micromol/l) for 2 min resulted in a similar transient platelet adhesion refractoriness to ECM under flow conditions. The transient platelet refractoriness and microaggregate formation induced by ADP was associated with a transient reduction in glycoprotein (GP)Ib, increased P-selectin expression and increased fibrinogen binding by circulating platelets. These data suggest a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus.
Global regulatory genes in Staphylococcus aureus, including agr and sar, are known to regulate the expression of multiple virulence factors, including cell wall adhesins. In the present study, the adherence of S. aureus RN6390 (wild type), RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) to immobilized fibrinogen, fibronectin, von Willebrand factor (vWF), extracellular matrix (ECM), and human endothelial cells (EC) EAhy.926 was studied. Bacteria grown to postexponential phase were subjected to light oscillation (static condition) or to shear stress at 200 s(-1) (flow condition) on tissue culture polystyrene plates coated with either protein ligands, ECM, or EC. Adherence of nonlabeled bacteria to immobilized ligands was measured by an image analysis system, while adherence of [(3)H]thymidine-labeled S. aureus to ECM and EC was measured by a beta-scintillation counter. The results showed increased adherence of agr and agr sar mutants to immobilized fibrinogen and higher potential of these mutants to induce platelet aggregation in suspension, decreased adherence of sar and agr sar mutants to immobilized fibronectin and vWF as well as to ECM and EC, increased adherence of both S. aureus wild type and sar mutant to EC treated with platelet-rich plasma (PRP) compared to platelet-poor plasma (PPP) and to EC treated with PPP compared to the control, and increased adherence of S. aureus wild type to EC coated with PRP in which platelets were activated with phorbol 12-myristate 13-acetate compared to intact PRP. This finding paralleled the increased adherence to EC of activated compared to intact platelets. It is suggested that platelet-mediated S. aureus adherence to EC depends on platelet activation and the number of adherent platelets and available receptors on the platelet membrane. In conclusion, the agr locus downregulates S. aureus adherence to fibrinogen, while the sar locus upregulates S. aureus adherence to fibronectin, vWF, ECM, and EC. The effect of both agr and sar on S. aureus adherence properties develops primarily under flow conditions, which suggests different adhesion mechanisms in static and flow conditions.
Recently, we described a method of testing platelet deposition on extracellular matrix under flow conditions. The method was used for assessment of platelet function in various platelet disorders, for monitoring of replacement and anti-platelet therapy. In the present study, we investigated platelet deposition on a polystyrene surface compared with that on extracellular matrix, under defined shear rates, using the original Cone and Plate(let) Analyzer. A correlation of adhesion rate (surface coverage) and aggregate formation (average size) of platelets from normal citrated blood between polystyrene and extracellular matrix was observed. Blocking of von Willebrand factor binding to glycoprotein Ib by a recombinant von Willebrand factor fragment substantially decreased platelet adhesion to both surfaces. Blocking of GPIIb-IIIa by Arg-Gly-Asp-Ser peptide prevented platelet adhesion to the polystyrene while an extensive adhesion of single platelets to extracellular matrix was observed. Furthermore, platelet adhesion to polystyrene but not to extracellular matrix was completely inhibited by platelet inactivation with prostaglandin E(1). Platelets from patients with severe von Willebrand disease yielded very low adhesion to both polystyrene and extracellular matrix. The addition of von Willebrand factor to the blood of these patients or pre-coating of polystyrene surface with von Willebrand factor restored the ability of platelets to adhere and aggregate on the surface. Platelets from patients with Glanzmann's thrombasthenia and afibrinogenemia adhered to extracellular matrix (with defective aggregate formation), while they failed to adhere to the polystyrene. Fibrinogen added to afibrinogenemia blood or pre-coating of the polystyrene with fibrinogen restored the ability of platelets to adhere and aggregate on the surface. In conclusion, the polystyrene surface, like extracellular matrix, can be used to assess platelet function disorders taking in account that platelet deposition on polystyrene under flow is absolutely dependent on platelet activation and on the presence of fibrinogen, von Willebrand factor, and their receptors.
The present study was performed to determine how sympathetic function is altered by simulated microgravity, dry immersion for 3 days, and to elucidate the mechanism of post-spaceflight orthostatic intolerance in humans. Six healthy men aged 21-36 years old participated in the study. Before and after the dry immersion, subjects performed head-up tilt (HUT) test to 30 degrees and 60 degrees (5 min each) with recordings of muscle sympathetic nerve activity (MSNA, by microneurography), electrocardiogram, and arterial blood pressure (Finapres). Resting MSNA was increased after dry immersion from 23.7+/-3.2 to 40.9+/-3.0 bursts/min (p<0.005) without significant changes in resting heart rate (HR). MSNA responsiveness to orthostasis showed no significant difference but HR response was significantly augmented after dry immersion (p<0. 005). A significant diastolic blood pressure fall at 5th min of 60 degrees HUT was observed in five orthostatic tolerant subjects despite enough MSNA discharge after dry immersion. A subject suffered from presyncope at 2 min after 60 degrees HUT. He showed gradual blood pressure fall 10 s after 60 degrees HUT with initially well-maintained MSNA response and then with a gradually attenuated MSNA, followed by a sudden MSNA withdrawal and abrupt blood pressure drop. In conclusion, dry immersion increased MSNA without changing MSNA response to orthostasis, and resting HR, while increasing the HR response to orthostasis. Analyses of MSNA and blood pressure changes in orthostatic tolerant subjects and a subject with presyncope suggested that not only insufficient vasoconstriction to sympathetic stimuli, but also a central mechanism to induce a sympathetic withdrawal might play a role in the development of orthostatic intolerance after microgravity exposure.
Recent studies emphasize the role of blood constituents in Staphylococcus aureus (S. aureus) adherence to subendothelial extracellular matrix. In the present study, the adherence of two strains of S. aureus (ATCC 29213 and RN 6390) grown to a postexponential phase to cultured human umbilical vein endothelial cells (EC-304) was examined. Under flow conditions (600 s(-1)), pretreatment of endothelial cells (ECs) with human alpha-thrombin (2 U/mL) significantly (2- to 4-fold) increased bacterial adherence to ECs. Adherence of both S. aureus strains to thrombin-treated ECs was similarly higher in the presence of whole blood, platelet-rich plasma, or platelet-poor plasma when compared with Tris-buffered saline solution (TBS). Platelet inactivation in whole blood by prostaglandin E1 did not reduce the adherence rate. When ATCC 29213 bacteria were suspended in TBS containing increasing concentrations of fibrinogen at near-physiologic ranges (0.25 to 2 mg/mL), a dose-dependent increase in S. aureus adherence to thrombin-activated ECs was observed that reached a maximum level of about 12-fold. Fibronectin used at the above physiologic concentrations (12.5 to 100 microg/mL) enhanced bacterial adherence up to 2-fold. Von Willebrand factor (1 IU/mL) did not support bacterial adherence to ECs, either alone or in combination with fibrinogen. Inhibition of fibrin formation either by the Gly-Pro-Arg-Pro peptide or by hirudin increased bacterial adherence by 50% and 90%, respectively. Blockage of either ICAM-I, alpha5beta1, or alphavbeta3 receptors on ECs by appropriate monoclonal antibodies resulted in substantial inhibition of bacterial adherence (by 42%, 65%, and 72%, respectively). Preincubation of S. aureus with a fibrinogen gamma-chain binding domain peptide led to 65% inhibition of adherence to ECs in the presence of fibrinogen. In contrast, preincubation of bacteria with the Arg-Gly-Asp-Ser peptide failed to affect their adherence. The data suggest that S. aureus adherence to the EC surface was (1) significantly enhanced by thrombin treatment of ECs, (2) not mediated by platelets, and (3) mediated by plasma fibrinogen, which binds to the bacteria via the C-terminus gamma-chain binding domain but not via the Arg-Gly-Asp sequence.
The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s-1) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.
We investigated the effects of two well established risk factors for cardiovascular disease, homocysteine and oxidized low density lipoprotein (ox-LDL), on endothelial cell thrombogenicity. For this purpose we studied platelet adhesion to human endothelial cells (EC) under flow conditions at a shear rate of 350 s(-1) following EC treatment with either homocysteine or ox-LDL. Treatment of EC with either homocysteine (1 or 10 mmol/L for 16 h) or ox-LDL (100 microg/ml for 16 h) resulted in a 2-3 fold enhancement in platelet adhesion. The enhancement in platelet adhesion induced by 1 mmol/L homocysteine, but not that induced by 10 mmol/L homocysteine, was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface tissue factor (TF) activity and slightly reduced the expression of the intercellular adhesion molecule I (ICAM-1). In contrast, ox-LDL treatment upregulated ICAM-1 expression and had no significant effect on endothelial TF activity. Neither homocysteine nor Ox-LDL affected surface expression of the alpha(v)beta3 integrin. The homocysteine-induced enhancement in platelet adhesion was almost completely abolished by blockade of the EC TF activity by a polyclonal antibody. The enhancing effect of homocysteine was also greatly reduced by inhibition of the EC alpha(v)beta3 integrin, but was not affected by blockade of EC ICAM-1. On the other hand, ox-LDL-induced enhancement in platelet - EC adhesion was greatly inhibited by blocking ICAM-1 or alpha(v)beta3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist Reo-Pro has virtually abolished the enhancing effect of both homocysteine and ox-LDL. Our results suggest that homocysteine and ox-LDL might increase endothelial thrombogenicity by distinct mechanisms: homocysteine - by inducing TF activity, and ox-LDL - by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen dependent platelet adhesion to EC. The alpha(v)beta3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation.
Antiplatelet drugs are the mainstays of therapy for acute and chronic cardiovascular diseases. S-nitroso-AR545C - an S-nitrosoderivative of a recombinant von Willebrand factor fragment AR545C spanning Ala 444 to Asp 730 and containing an Arg 545 Cys mutation, was previously found to inhibit ristocetin- and ADP-induced platelet aggregation and the interaction of platelets with extracellular matrix (ECM). In the current study we tested the antithrombotic properties of S-nitroso-AR545C on guinea pig platelets and in a platelet-rich thrombosis model in the guinea pig. Preincubation of guinea pig platelets with 0.1 microM of S-nitroso-AR545C decreased ristocetin-induced agglutination by 40% (p = 0.009) and completely abolished ADP-induced aggregation (p <0.0001). At concentration of 1.0 microM, S-nitroso-AR545C completely inhibited platelet adhesion (represented by surface coverage - SC) and decreased aggregate formation (represented by average aggregate size - AS) by more than 50%. Treatment of guinea pigs with 1.0 mg/kg S-nitroso-AR545C resulted in a significantly delayed time to arterial occlusion (31.7+/-6.0 min vs. 13.9+/-3.2 min, p <0.02). Similarly, total patency time was longer in the group injected with S-nitroso-AR545C compared to the control group. However, the difference was not statistically significant (33.8+/-6.3 min vs. 20.2+/-3.3 min, p = 0.07). No change in platelet count, hematocrit and bleeding time was observed 60 min after injection compared to baseline. In contrast, a significant decrease in SC (p <0.0001) and AS (p <0.01) were observed 60 min after the injection of S-nitroso-AR545C, whereas no change in these parameters was observed in the control group. These observations indicate that S-nitroso-AR545C exhibits significant antiadhesive and antiaggregating effects in-vitro and inhibits clot formation in-vivo suggesting that this compound may have potential therapeutic advantages.
We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel < > appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.
In a previous report by Freedman et al (J Clin Invest. 1996;97:979-987), plasma from 2 brothers with stroke or transient ischemic attack inactivated the antiplatelet effects of nitric oxide (NO), and this effect was found to be a consequence of a deficiency of plasma glutathione peroxidase (GSH-Px). In this study, we attempted to define the generalizability of this deficiency by studying NO-mediated antiplatelet effects in 7 families with familial childhood stroke. Seven families with familial childhood stroke that consecutively presented to a large referral center were included in the study. We monitored ADP-induced aggregation of normal gel-filtered platelets (GFP) in platelet-poor plasma (PPP) from normal individuals and from patients in the presence or absence of an NO donor (S-nitroso-glutathione). Surface P-selectin expression of normal GFP in patients' PPP was analyzed by flow cytometry after incubation with a P-selectin-specific monoclonal antibody in the presence or absence of the NO donor. We also measured GSH-Px activity in plasmas from family members and normal controls using standard methods. In 6 of 7 families, NO failed to inhibit platelet P-selectin expression and platelet aggregation in PPP from the affected family members and some of their relatives. Of 4 families studied, 3 probands and their corresponding affected parent had 50% decrease in plasma GSH-Px activity. In some patients with childhood stroke, impaired metabolism of reactive oxygen species as a result of reduced GSH-Px activity results in NO insufficiency that affects normal platelet inhibitory mechanisms and predisposes to arterial thrombosis.
In vitro platelet function of umbilical cord blood and neonatal peripheral vein blood from full-term newborns was compared with that of adults. Citrated whole blood was subjected to shear stress (1300 s(-1)) on subendothelial extracellular matrix (ECM)-coated wells in a cone and plate(let) analyzer. Adhered platelets on the ECM were quantitated by image analyzer. Both umbilical cord and neonatal peripheral blood platelets demonstrated more extensive adhesion than adult platelets, and similar aggregate formation on ECM. The ability of neonatal platelets to form aggregates on ECM was confirmed by scanning electron microscopy. Similar activation of neonatal and adult platelets after subjection to shear stress, in the suspension phase, was established by flow cytometry, which showed an increase in fibrinogen binding and a decrease in glycoprotein Ib expression on platelet membrane. The difference in adhesion rates between neonatal and adult platelets was preserved even when the hematocrit level of the neonatal blood was adjusted to that of adults. Reconstitution of neonatal or adult platelet-rich plasma with autologous or heterologous red packed cells yielded no change in adhesion and aggregation. When von Willebrand factor-covered plates were used to prevent deposition of plasma von Willebrand factor on the surface, no difference in platelet adhesion was seen between neonatal and adult blood. In conventional aggregometry assay, the response to ristocetin of washed platelets of either neonatal or adult source was higher on addition of plasma from neonates than from adults. Our data suggest that the extensive neonatal platelet deposition on ECM is mediated by plasma von Willebrand factor, which is known to be more multimerized and, therefore, more active in neonates than in adults. This mechanism may provide balanced primary hemostasis in neonates despite the platelet hyporeactivity to agonists without application of shear stress.
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Increased platelet aggregation has been suggested to play a role in the accelerated atherosclerosis of diabetics. However the physiological relevance of the aggregation tests has been questioned. The purpose of this study was to determine platelet activation in diabetic patients, using a novel device--the cone and plate(let) analyzer--to measure shear-induced platelet adhesion and aggregation on extracellular matrix (ECM). Whole blood platelet adhesion and aggregation in patients with noninsulin-dependent diabetes mellitus (n=82) and in nondiabetic controls (n=71) were compared. Clinical and laboratory characteristics of the diabetic patients were analyzed for possible correlation with parameters of platelet activity. Patients with diabetes had a significantly increased platelet activation compared to nondiabetic subjects, demonstrated by an increased adhesion to the ECM (surface coverage, 23% [95% confidence interval, 22-25%] vs. 19% [95% confidence interval, 18-20%], respectively) and an increased average size of the ECM-bound aggregates (54 microm2 [95% confidence interval, 51-57 microm2] vs. 47 microm2 [95% confidence interval, 43-51 microm2], respectively). Platelet adhesion in the diabetic group was found to correlate with triglyceride levels (r=0.36) and hematocrit values (r=0.31) and inversely with high-density lipoprotein cholesterol levels (r=0.30). There were no correlation, however, between parameters of platelet reactivity and duration of diabetes, vascular complications and low-density lipoprotein levels. Our data demonstrate an increased platelet adhesion and aggregation in diabetic patients and suggest a modulatory role of diabetic dyslipidemia.
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the beta 1 and beta 2 integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti-PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s-1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation resonses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the alpha IIb beta 3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.
The risk of bleeding among thrombocytopenic patients was evaluated using our new cone and platelet analyser (CPA) test. Using this test, adherence of platelets was quantitated on extracellular matrix and expressed as percent of surface coverage (SC) and the average size (AS) of aggregates. 42 thrombocytopenic patients with ITP (n=23), post chemotherapy (n= 12) and others (n= 7) were tested over a total of 82 visits. On each visit, complete blood count and CPA tests were performed and patients were evaluated for evidence of bleeding (found in 40 visits). Bleeding patients had significantly lower platelet counts (27.4 +/- 22.0 v 47.1 +/- 21.0 x 10(9)/l), lower haematocrit values (30.2 +/- 8.1 v 35.2 +/- 6.6%), lower MPV (6.83 +/-1.89 v 8.98 +/- 1.13 fl), and lower SC (4.87 +/- 3.95 v 10.33 +/-5.48%) and AS (33.99 +/- 14.94 v 52.9 +/- 24.34 microm2). Univariate analysis yielded platelet count < or =20.0 x 10(9)/l, MPV < or =8 fl, haematocrit <35%, SC <5%, AS< or =40 microm2 as significantly associated with bleeding, whereas only MPV and SC were associated with bleeding (OR 6.95, CI 2.25-21.46 and OR 4.27, CI 1.29-14.16, respectively) by multivariate analysis. When taken together, 21/22 of patients (95%) with both low SC (<5%) and low MPV (<8.0 fl) had bleeding symptoms, whereas only 9/43 (21%) patients with both these parameters above these values experienced bleeding symptoms. We conclude that the CPA test and the parameter SC (<5%) together with MPV (< or =8 fl) might be used as independent predictors of bleeding in the management of thrombocytopenic patients.
We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post-transplant and declined after 6 weeks, while human anti-human platelet antibodies were detected 2-8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross-reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP.
Platelet adhesion to exposed subendothelium is mediated by platelet receptor glycoprotein Ib and polymeric von Willebrand factor (vWF). To improve the results of coronary arterial thrombolysis, fragments of vWF with enhanced glycoprotein Ib binding competitive with native vWF have been proposed as adjuvants to recombinant tissue-type plasminogen activator (rtPA). We designed a recombinant vWF fragment spanning Ala444 to Asp730 that contains the Arg545Cys mutation (named AR545C) and analyzed its antiplatelet properties in vitro and in vivo. AR545C-platelet interaction was assessed by ristocetin or botrocetin-induced platelet agglutination, or interaction with extracellular matrix under arterial flow conditions. AR545C showed enhanced reactivity with platelet glycoprotein Ib at low concentrations of ristocetin, and 60% bound spontaneously to platelets. AR545C inhibited ristocetin-induced platelet agglutination in a dose-dependent manner, with a concentration necessary to inhibit 50% of agglutination of 0.16+/-0.04 micromol/L. The inhibitory effect of AR545C on rabbit botrocetin-induced platelet agglutination was also dose dependent, with a concentration necessary to inhibit 50% of agglutination of 0.3 to 0.5 micromol/L. AR545C also completely inhibited aggregate formation and decreased the adhesion of platelets to extracellular matrix by 62.5%. The effect of AR545C on thrombolysis with rtPA was evaluated using a modified rabbit femoral thrombosis model. Local injection of AR545C into the thrombosed segment of rabbit femoral artery significantly shortened the time to reperfusion with rtPA (60+/-17.3 versus 103+/-15.2 minutes, P=.05) and significantly prolonged the total patency time (175 versus 21 minutes, P=.04). No significant difference was found in the reperfusion rate or time to reocclusion. AR545C is a potential antithrombotic agent that enhances the thrombolytic effect of rtPA in the rabbit model.