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R Dardik

Publications and source records attributed to R Dardik.

At least 19 recordsLinked to original sources
PubMed2002

Deposition of whole blood platelets on extracellular matrix under flow conditions in preterm infants.

BACKGROUND: A previous study showed greater adhesion by platelets of healthy full term infants to subendothelial extracellular matrix (ECM) under flow conditions compared with healthy adult platelets. AIM: To investigate the adhesion and aggregation of platelets from preterm infants on ECM under defined shear conditions. METHODS: In vitro platelet function was investigated in 106 preterm infants, 74 full term infants, and 26 healthy adults. Blood samples were obtained from all infants within 24 hours of birth, and weekly until discharge from preterm infants only. Citrated whole blood was placed in ECM precoated tissue culture plates and subjected to shear stress (1300 s-1) for two minutes using a rotating Teflon cone. Platelet adhesion (surface coverage) and aggregation (average size) to ECM were assayed using an image analyser. Assays for von Willebrand factor (vWF) antigen, ristocetin cofactor, and vWF collagen-binding activity were performed on samples from an additional 70 preterm infants, 23 healthy full term infants, and 24 healthy adults. Preterm infants with hyaline membrane disease (HMD) were analysed separately in both cohorts. RESULTS: Platelets from preterm infants displayed significantly less platelet adhesion than those from full term infants but similar aggregation and levels of vWF antigen, ristocetin cofactor, and collagen binding activity. Mean surface coverage was 22.0 (8.4)% for preterm infants with HMD, 28.7 (8.0)% for healthy preterm infants, and 35.7 (7.9)% for full term infants. Surface coverage in the preterm infants correlated with gestational age during the first 24 hours only, and did not reach full term levels during 10 weeks of follow up. CONCLUSION: Platelet adhesion to ECM is significantly poorer in preterm than in full term infants, and poorer in preterm infants with HMD than in healthy preterm infants. Intrinsic platelet properties rather than the concentration or activity of vWF may be responsible for this difference.

Adult↗
PubMed2001

Cerebrovascular events in patients with significant stenosis of the carotid artery are associated with hyperhomocysteinemia and platelet antigen-1 (Leu33Pro) polymorphism.

BACKGROUND AND PURPOSE: Although risk factors for carotid artery stenosis caused by atherosclerosis are known, it is unclear what triggers "activation" of the atherosclerotic plaques and the ensuing thromboembolic cerebral events. The aim of this study was to evaluate whether thrombophilic factors, platelet glycoprotein (GP) polymorphisms, and homocysteine are associated with a risk of ischemic events in patients with significant carotid stenosis. METHODS: Consecutive patients with >/=50% carotid stenosis, whether symptomatic (with ipsilateral ischemic events) or asymptomatic, who were evaluated and followed in a neurovascular clinic were tested for plasma levels of homocysteine, C677T mutation in methylenetetrahydrofolate reductase, G20210A mutation of factor II, factor V Leiden, antiphospholipid antibodies, and polymorphisms of platelet membrane GP: human platelet antigen (HPA)-1, GP Ia (C807T), and GP Ib (variable number of tandem repeats, Kozak, and HPA-2). RESULTS: Eighty-six asymptomatic and 67 symptomatic patients were evaluated. The former group was older (73.7+/-6.9 versus 69.5+/-9.1 years, P=0.02). Major risk factors for stroke were similar in both groups. In symptomatic patients versus asymptomatic patients, hyperhomocysteinemia was 3-fold more frequent (34.3% versus 12.8%, respectively; P=0.002) and HPA-1a/b was almost 2-fold more common (38.8% versus 20.9%, respectively; P=0.01). All other thrombophilic factors and platelet polymorphisms studied did not differ significantly between the 2 groups. Multivariate analysis revealed that hyperhomocysteinemia and the HPA-1a/b genotype conferred a significant risk of cerebral ischemic events, with odds ratios (95% CI) of 4.07 (1.7 to 9.7) and 3.4 (1.5 to 7.8), respectively. CONCLUSIONS: Hyperhomocysteinemia and HPA-1a/b are independent risk factors for ischemic events in patients with significant carotid stenosis.

Aged↗
PubMed2001

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia.

The most frequent mutation causing Glanzmann thrombasthenia in Iraqi-Jews (IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. This deletion predicts a frameshift that results in the elimination of the C406-C655 disulfide bond and a premature termination codon shortly before the transmembrane domain. To determine the contribution of each of these alterations to the thrombasthenic phenotype, Chinese hamster ovary or baby hamster kidney cells were cotransfected with normal GPIIb complementary DNA (cDNA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 2011T>A mutated cDNA predicting C655S (single-letter amino acid codes) substitution, and 2019A>T mutated cDNA predicting Stop657. Elimination of the C406-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surface expression or binding of the transfected cells to immobilized fibrinogen, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the transfected cells to immobilized fibrinogen. Immunohistochemical staining and immunoprecipitation demonstrated that the elimination of amino acids 657-762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formation, and differential immunofluorescence staining of GPIIIa and cellular organelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower efficiency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa. (Blood. 2001;98:1063-1069)

Animals↗
PubMed2001

Methionine synthase A2756G and methylenetetrahydrofolate reductase A1298C polymorphisms are not risk factors for idiopathic venous thromboembolism.

Hyperhomocysteinemia is a defined risk factor for venous thromboembolism (VTE). Several polymorphisms of genes encoding for enzymes acting in the remethylation pathway of homocysteine metabolism, ie, methionine synthase (MS) A2756G, methylenetetrahydrofolate reductase (MTHFR) C677T and MTHFR A1298C, can cause increased homocysteine levels particularly in patients with deficiencies of folic acid, vitamin B6, or B12 and hence be potential risk factors for VTE. Indeed, homozygous MTHFR C677T was shown to be a mild risk factor for VTE by some, but not by all, investigators. In this study, we assessed the risk exerted by MS A2756G and MTHFR A1298C in a cohort of patients with idiopathic venous thromboembolism. Homozygosities for MS A2756G and MTHFR A1298C were not found to be statistically significant risk factors for VTE. In addition, no interactions were observed among MS A2756G, MTHFR A1298C and MTHFR C677T in conferring a risk of VTE.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2001

Transient adhesion refractoriness of circulating platelets under shear stress: the role of partial activation and microaggregate formation by suboptimal ADP concentration.

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.

Adenosine Diphosphate↗
PubMed2001

Differential expression and regulation of CD6 on T-cell subsets revealed by monoclonal antibody (MAb) CH11.

A monoclonal antibody (MAb), CH11, was developed by immunizing mice with CD4+ gammadelta T-cell receptor (TCR)+ cells. It recognized an antigen expressed in the surface membrane of T-cell lines, but not of U937, lymphoblastoid B cells (LBC), K562, Raji or Daudi cells, indicating selectivity for the T-cell lineage. In addition, it labelled 70-80% of normal peripheral blood mononuclear cells (PBMC), with high expression on the erythrocyte rosetting (E+) fraction, and low/absent expression on E- cells. However, CD4+ T cells expressed higher levels of reactivity than CD8+ or gammadelta+ T-cell receptor (TCR)+ lymphocytes in PB. Furthermore, in 7 of 10 individuals tested, 7.34+/-3.88% of unselected PBMC were CH11- CD3+ and were relatively enriched in CD8+ and in gammadelta TCR+-cells. In addition, thymic gammadelta T cells, and gammadelta lymphoproliferations from two patients were nonreactive or weakly reactive with the MAb. Activation of E+ cells with phorbol-12-myristate-13-acetate (PMA) enhanced CH11 expression uniformly, whereas activation with phytohemagglutinin (PHA) selectively down-regulated expression of the antigen on the CD8+ subset. In Western blots performed in nonreducing (NR) conditions, MAb CH11 detected a 100 kDa molecule in PBMC and Jurkat T-cell lysates. Preincubation of T cells with MAb CH11 specifically abrogated their subsequent reactivity with MAb to CD6, suggesting that MAb CH11 is recognizing an epitope of CD6. Given its function as a receptor for ligands on thymic epithelium, activated leukocytes and synoviocytes, this newly defined heterogeneity of expression and regulation of the CD6 molecule on subsets of T cells may help determine their functional repertoire in vivo.

Animals↗
PubMed2001

Adherence properties of Staphylococcus aureus under static and flow conditions: roles of agr and sar loci, platelets, and plasma ligands.

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.

Animals↗
PubMed2000

Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor.

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.

Afibrinogenemia↗
PubMed2000

Staphylococcus aureus adherence to thrombin-treated endothelial cells is mediated by fibrinogen but not by platelets.

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.

Alprostadil↗
PubMed2000

The role of angiotensin converting enzyme and angiotensin II type 1 receptor gene polymorphisms in patients with nonarteritic anterior ischemic optic neuropathy.

OBJECTIVE: To determine the role of angiotensin converting enzyme (ACE) and angiotensin II type 1 receptor (AT1R) polymorphisms in the pathogenesis of nonartertic anterior ischemic optic neuropathy (NAION). DESIGN: Retrospective, case-control study. PARTICIPANTS: Seventy-four patients with NAION diagnosed from 1984 through 1999. Seventy-one patients who visited the Eye Institute comprised the control group. TESTING INTERVENTION: DNA was extracted from whole blood obtained from all patients and control participants. Polymerase chain reaction (PCR) was used for analysis of ACE and AT1R polymorphisms. RESULTS: The frequency of the polymorphism for ACE among the NAION patients (39.2% deletion allele [DD], 54.0% deletion/insertion [D/I] locus, 6.8% insertion allele [II]) was similar to that of the control group (50.7% DD, 39.4% D/I, 9.9% II), with P = 0.21. The frequency of the polymorphism of AT1R in the NAION patients was 5.4% CC, 44.6% CA, 50% AA, and in the control group it was 4.2% CC, 33.8% CA, 62.0% AA, with P = 0.35. Participants less than 55 years of age and those more than 55 had quite similar distributions. CONCLUSIONS: Angiotensin converting enzyme and AT1R polymorphisms have no part in the mechanism of NAION. Thus drugs such as ACE inhibitors or AT1R antagonists are not specifically indicated for treatment of these patients.

Adult↗
PubMed2000

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells.

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).

Blood Platelets↗
PubMed2000

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP.

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.

Blood↗
PubMed2000

Homocysteine and oxidized low density lipoprotein enhanced platelet adhesion to endothelial cells under flow conditions: distinct mechanisms of thrombogenic modulation.

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.

Abciximab↗
PubMed1999

Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin.

Thrombospondin-1 (TSP-1) interacts specifically with heparin and fibronectin in vitro and colocalizes with fibronectin and heparan sulfate in the extracellular matrix (ECM). Its conformation is strongly dependent on Ca2+ concentration. We have previously shown that both heparin and fibronectin have two binding sites on the TSP-1 subunit which may require conformational change for their occupancy (R. Dardik and J. Lahav, 1987, Eur. J. Biochem. 168, 347; ibid 1989, 185, 581). To investigate the effect of TSP-1 binding to fibronectin and heparin on its functional conformation, TSP-1 was subjected to proteolysis in the presence and absence of ligands and of Ca2+. We found that while trypsin cleavage of free TSP-1 resulted in the inactivation of ligand binding, TSP-1 bound to either fibronectin or heparin remained stably associated with these ligands. Cleavage by thrombin or tissue plasminogen activator (tPA) showed that Ca2+-depleted TSP-1, when bound to fibronectin or to heparin, yielded proteolytic cleavage patterns typical of the Ca2+-containing form. Cleavage by chymotrypsin was not affected by binding to fibronectin or heparin; hence loss of proteolytic susceptibility was not due to steric hindrance by the ligands. Taken together, these results indicate that: (A) binding of TSP-1 to fibronectin or heparin is a two-step mechanism where binding to one site leads to conformational changes that enable binding to the second site; (B) TSP-1 in complex with fibronectin or heparin adopts the Ca2+-containing conformation in the absence of Ca2+; and (C) such complexes are highly resistant to cleavage by tPA and, if cleaved by other enzymes, the TSP-1 fragments remain bound to other ECM components. These characteristics have profound significance for platelet adhesion and cell migration into wounds where Ca2+ concentrations are reduced.

Animals↗
PubMed1999

Synergistic effects of prothrombotic polymorphisms and atherogenic factors on the risk of myocardial infarction in young males.

Several recent studies evaluated a possible effect of the prothrombotic polymorphisms such as 5,10 methylenetetrahydrofolate reductase (MTHFR) nt 677C --> T, factor V (F V) nt 1691G --> A (F V Leiden), and factor II (F II) nt 20210 G --> A on the risk of myocardial infarction. In the present study, we analyzed the effect of these prothrombotic polymorphisms, as well as apolipoprotein (Apo) E4, smoking, hypertension, diabetes mellitus, and hypercholesterolemia, on the risk of myocardial infarction in young males. We conducted a case-control study of 112 young males with first acute myocardial infarction (AMI) before the age of 52 and 187 healthy controls of similar age. The prevalences of heterozygotes for F V G1691A and F II G20210A were not significantly different between cases and controls (6.3% v 6.4% and 5.9% v 3.4% among cases and controls, respectively). In contrast, the prevalence of MTHFR 677T homozygosity and the allele frequency of Apo E4 were significantly higher among patients (24.1% v 10.7% and 9.4% v 5.3% among cases and controls, respectively). Concomitant presence of hypertension, hypercholesterolemia, or diabetes and one or more of the four examined polymorphisms increased the risk by almost ninefold (odds ratio [OR] = 8.66; 95% confidence interval [CI], 3.49 to 21.5) and concomitant smoking by almost 18-fold (OR = 17.6; 95% CI, 6.30 to 48.9). When all atherogenic risk factors were analyzed simultaneously by a logistic model, the combination of prothrombotic and Apo E4 polymorphisms with current smoking increased the risk 25-fold (OR = 24.7; 95% CI, 7.17 to 84.9). The presented data suggest a synergistic effect between atherogenic and thrombogenic risk factors in the pathogenesis of AMI, as was recently found in a similar cohort of women.

Activated Protein C Resistance↗
PubMed1999

Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism: prevalence and risk assessment.

The inherited thrombophilias--deficiencies of protein C, protein S, and antithrombin III--and the prothrombotic polymorphisms factor V G1691A and factor II G20210A predispose patients toward venous thromboembolism (VTE). The aim of this study was to determine the prevalence of single and combined prothrombotic factors in patients with idiopathic VTE and to estimate the associated risks. The study group consisted of 162 patients referred for work-up of thrombophilia after documented VTE. The controls were 336 consecutively admitted patients. In all subjects factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were analyzed by specific polymerase chain reactions and restriction enzymes. Activities of antithrombin III and protein C, free protein S antigen, and lupus anticoagulant were determined in a subset of 109 patients who were not receiving oral anticoagulants. The prevalences of heterozygotes and homozygotes for factor V G1691A and factor II G20210A among patients and controls were 40.1% versus 3.9% and 18.5% versus 5.4%, respectively (P=0.0001). The prevalence of homozygotes for MTHFR C677T in patients was 22.8% and in controls, 14.3% (P=0.025). Heterozygous and homozygous factor V G1691A, factor II G20210A, and homozygous MTHFR C677T were found to be independent risk factors for VTE, with odds ratios of 16.3, 3.6, and 2.1, respectively. Two or more polymorphisms were detected in 27 of 162 patients (16.7%) and in 3 of 336 controls (0.9%). Logistic regression analysis disclosed odds ratios of 58.6 (confidence interval [CI], 22.1 to 155.2) for joint occurrence of factor V and factor II polymorphisms, of 35.0 (CI, 14.5 to 84.7) for factor V and MTHFR polymorphisms, and of 7.7 (CI, 3.0 to 19.6) for factor II and MTHFR polymorphisms. Among 109 patients in whom a complete thrombophilic work-up was performed, 74% had at least 1 underlying defect. These data indicate that in most patients referred for evaluation of thrombophilia due to idiopathic VTE, 1 or more underlying genetic predispositions were discernible. The presence of >1 of the prothrombotic polymorphisms was associated with a substantial risk of VTE.

Adult↗
PubMed1999

Increased neonatal platelet deposition on subendothelium under flow conditions: the role of plasma von Willebrand factor.

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.

Adult↗