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Biomedical subjects

B S Coller

Publications and source records attributed to B S Coller.

At least 91 records · Page 5Linked to original sources
PubMed1991

A deletion in the gene for glycoprotein IIb associated with Glanzmann's thrombasthenia.

The platelet fibrinogen receptor is composed of a complex of glycoproteins (GP) IIb and IIIa on the surface of platelets. Deficient function of this receptor prevents normal platelet aggregation, resulting in Glanzmann's thrombasthenia (GT). In this paper, we describe a black thrombasthenic patient who is either homozygous or hemizygous for a deletion within the GPIIb gene. Initial Western blot analysis of platelet proteins from this patient did not detect any GPIIb, but did detect small amounts of GPIIIa of normal mobility. Quantitation of vitronectin receptor (VNR) demonstrated that this thrombasthenic patient had approximately 1.5-2 times the number of these receptors per platelet compared with controls, a finding that has previously been noted in other thrombasthenic patients with defects in GPIIb. Genomic Southern blot studies demonstrated a deletion in the GPIIb gene of approximately 4.5 kilobasepairs (kb). Analysis of the isolated GPIIb gene demonstrated that the deletion begins between two Alu repeats within intron 1 and ends in intron 9. Polymerase chain reaction (PCR) studies using platelet RNA and oligonucleotides directed to both the 5' and 3' ends of the GPIIb cDNA sequence easily detected GPIIb transcript, suggesting that the genomic deletion of exons 2-9 does not significantly decrease the level of the GPIIb mRNA. Sequence analysis of PCR-generated GPIIb cDNA showed that a cryptic AG splice acceptor sequence was being utilized, resulting in a transcript that contained a portion of introns 1 and 9, as well as having a deletion of exons 2-9. Unlike the GPIIb gene, the GPIIIa gene appears to be intact by Southern blot analysis. PCR studies using platelet RNA and oligonucleotides directed to the GPIIIa cDNA sequence demonstrated the presence of GPIIIa mRNA. In summary, the thrombasthenic state in this patient appears to be due to a GPIIb gene deletion resulting in an abnormal transcript and no detectable platelet GPIIb. Platelet GPIIIa levels were secondarily low presumably due to the known instability of GPIIIa in the absence of GPIIb.

Adolescent↗
PubMed1990

Pharmacodynamic study of F(ab')2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein IIb/IIIa in patients with unstable angina pectoris.

The pharmacodynamics of intravenous bolus injections of 0.05, 0.10, 0.15, and 0.20 mg/kg of F(ab')2 fragments of the murine monoclonal antibody 7E3, 7E3-F(ab')2, directed against the glycoprotein IIb/IIIa (GPIIb/IIIa) receptor of human platelets, were studied in groups of four patients with unstable angina pectoris. With 0.20 mg/kg, the template bleeding time prolonged from 6.3 +/- 1.9 (mean +/- SD) to greater than 30 min; it subsequently decreased to 13 +/- 7.8 min after 12 h and to 8.3 +/- 1.5 min after 24 h. The number of unblocked GPIIb/IIIa receptors (preinfusion value, 32,000 +/- 3,000 per platelet) decreased to 13 +/- 7% of the preinfusion value 1 h after infusion, and then increased to 33 +/- 10% at 12 h, 44 +/- 8% at 24 h and 67 +/- 7% at 72 h. The logarithm of the bleeding time was inversely proportional with the residual GPIIb/IIIa receptors (r = 0.73, P less than 0.0001). ADP-induced platelet aggregation (measured by changes in light transmittance in percent) decreased from 60 +/- 5% before infusion to 1.5 +/- 3% 1 h after infusion; it then increased to 29 +/- 3% after 24 h and 39 +/- 6% after 72 h. Platelet counts decreased by 16% at 1 h and returned to control values within 24 h. Proportionally smaller effects were seen at lower doses of 7E3-F(ab')2. Antibody injection did not induce spontaneous bleeding. Angina was not observed during the first 12 h when the bleeding time was significantly prolonged, but occurred in 6 of the 16 patients within the next 3 d. 2 of the 16 patients developed low titers of IgG antibodies specific for 7E3-F(ab')2. Thus 7E3-F(ab')2 induces dose-related inhibition of platelet function; at a dose of 0.20 mg/kg, it causes profound inhibition of platelet aggregation and prolongation of the bleeding time, but no spontaneous bleeding.

Adult↗
PubMed1989

Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage.

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.

Blood Platelets↗
PubMed1989

Immunoblot analysis of platelet glycoprotein IIb in patients with Glanzmann thrombasthenia in Israel.

Recent studies have indicated that severe ('type I') Glanzmann thrombasthenia is a heterogeneous hereditary disorder caused by quantitative and/or qualitative abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Immunoblot analysis of sodium dodecyl sulphate (SDS)-solubilized platelets was carried out on controls and 18 patients (12 Iraqi-Jews, two Iranian Jews and four Arabs) employing three antibodies (one monoclonal and two polyclonal) directed at different sites on GPIIb. Nonreduced control platelet samples contained a major Mr approximately 140k immunoreactive protein that was split into an Mr approximately 120k (alpha) and an Mr approximately 25k (beta) band after reduction with mercaptoethanol. The nonreduced samples from all 18 patients tested had trace amounts of Mr approximately 140k band corresponding to normal GPIIb; the intensity of this band was estimated to be less than 1% of the normal amount. Unlike the control samples, however, this Mr approximately 140k band did not change electrophoretic mobility following reduction. Since GPIIb originates from a single chain precursor molecule of Mr approximately 140k that comprises both the alpha and beta chains, and which does not change mobility with reduction, our data suggest that the platelets of these patients contain small amounts of this precursor.

Antibodies, Monoclonal↗
PubMed1989

Correlation between template bleeding times and spontaneous bleeding during treatment of acute myocardial infarction with recombinant tissue-type plasminogen activator.

The purpose of this study was to correlate bleeding complications during and after treatment with recombinant tissue-type plasminogen activator (rt-PA) with serial template bleeding time measurements, with ADP-induced platelet aggregation, with clinical characteristics, and with hemostatic parameters. Fifty-two of 55 consecutive patients with acute myocardial infarction and template bleeding times (Ivy method) of less than 9.5 minutes were treated with rt-PA in a total dose of 55-212 mg (mean, 109 mg) over 90 to 360 minutes (median, 240 minutes) combined with heparin. The mean bleeding time was significantly prolonged at 90 minutes (from 5.0 +/- 1.9 to 8.2 +/- 4.3 minutes, p less than 0.0001) but returned toward baseline after 4 hours (from a median of 8.0 to 7.0 minutes, p less than 0.05). Thirteen patients (25%) suffered relatively minor but spontaneous bleeding that did not correlate with age, hypertension, smoking, partial thromboplastin time, platelet count, ADP-induced platelet aggregation, steady-state rt-PA level, or extent of fibrinogen degradation. In multivariate analysis, only the 90-minute bleeding time correlated with spontaneous bleeding (p = 0.01). Prolongation of the 90-minute bleeding time to greater than or equal to 9 minutes, which occurred in 21 patients, correlated with spontaneous bleeding with a sensitivity of 69% (95% confidence interval, 39-90%) and a specificity of 69% (95% confidence interval, 52-83%). Retrospective analysis revealed that in 14 patients taking aspirin, the bleeding time at 90 minutes was significantly more prolonged (p less than 0.05) and spontaneous bleeding significantly more frequent (p less than 0.01) than in patients not taking aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1989

Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIIb/IIIa receptor. Correlation with bleeding time, platelet aggregation, and blockade of GPIIb/IIIa receptors.

We studied the dose-response effects of the F(ab')2 fragments of murine monoclonal antibodies to the platelet GPIIb/IIIa receptor (7E3 and 10E5) on in vivo platelet thrombus formation in a well-characterized monkey model in which the carotid artery is stenosed and thrombus formation is provoked and augmented by intimal damage and the infusion of subaggregating doses of epinephrine. Both antibodies abolished thrombus formation with a mean dose of -0.2 mg/kg. Ex vivo platelet aggregation was not always abolished at doses that abolished thrombus formation; similarly, bleeding times were only moderately prolonged (9.1 +/- 1.4 minutes) at these doses. Increasing the dose above that required to abolish thrombus formation consistently produced abolition of ex vivo platelet aggregation, marked prolongation of the bleeding time (14.2 +/- 1.5 minutes), and nearly quantitative blockade of GPIIb/IIIa receptors. We conclude that in a significant percentage of animals, the extent of GPIIb/IIIa blockade required to prevent vasoocclusive thrombus formation in this model is less than that required for abolition of platelet aggregation, and that the preservation of only a minority of functional GPIIb/IIIa receptors might be adequate to maintain a nearly normal bleeding time.

Animals↗
PubMed1989

Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins.

Using intact human platelets as the immunogen and a functional, collagen-coated bead agglutination assay, we have produced a murine monoclonal antibody (6F1) that blocks the interaction between platelets and collagen in the presence of Mg++. 6F1 affinity-purified the platelet glycoprotein Ia/IIa complex, and approximately 800 molecules of 6F1 bound per platelet at saturation. 6F1 nearly completely inhibited collagen-induced platelet aggregation and inhibited platelet adhesion to collagen by greater than 95% when plasma proteins were absent. Antibody 10E5, which blocks the binding of adhesive glycoproteins to GPIIb/IIIa, produced only minor inhibition (approximately 25%) of adhesion under the same circumstances. In contrast, when tested in platelet-rich plasma (PRP), 6F1 had only a minor effect on collagen-induced platelet aggregation, prolonging the lag phase but not the slope or maximum aggregation. Similarly, when collagen was precoated with plasma, 6F1 caused less inhibition of platelet adhesion (53%) than without the precoating (greater than 95%). Antibody 10E5 inhibited this adhesion by 32%, and the combination of 6F1 and 10E5 was more effective than either alone, inhibiting it by 90%. Time course studies of platelet agglutination of collagen-coated beads using PRP containing physiologic concentrations of divalent cations showed early inhibition by 6F1, indicating that the GPIa/IIa receptor operates in this environment. With more prolonged incubation, however, 6F1 was less effective; this later agglutination could be partially prevented by adding 10E5 or PGE1 to the 6F1. These data support a model wherein collagen can directly interact with GPIa/IIa and can indirectly interact with GPIIb/IIIa via intermediary adhesive proteins. The physiological significance of these interactions, and potential interactions with other receptors, remains to be established.

Antibodies, Monoclonal↗
PubMed1988

Inhibition of human platelet function in vivo with a monoclonal antibody. With observations on the newly dead as experimental subjects.

The F(ab')2 fragment of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor (7E3) is a potent inhibitor of both in-vitro platelet aggregation and in-vivo platelet thrombus formation in animal studies. As a first step in assessing the potential of 7E3-F(ab')2 as an antithrombotic agent for use in humans, we administered 7E3-F(ab')2 intravenously at increasing doses to a person who had just died and was being maintained on a respirator (neomort). At 0.1 and at 0.2 mg/kg body weight, 74% and 92% of the glycoprotein IIb/IIIa receptors were blocked, respectively; adenosine-diphosphate-induced platelet aggregation was inhibited by 84% and 100% at these same doses. Platelet glycoprotein Ib function remained intact, even at 0.6 mg/kg. Acute hemodynamic or hemorrhagic toxicity was not noted. This antibody fragment, a potent, immediate-acting inhibitor of platelet aggregation, may be of benefit in vaso-occlusive and thromboembolic disorders.

Adenosine Diphosphate↗
PubMed1988

Rapid and sustained coronary artery recanalization with combined bolus injection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet GPIIb/IIIa antibody in a canine preparation.

The effects of bolus injections of recombinant single-chain tissue-type plasminogen activator (rt-PA) and of F(ab')2 fragments of a murine monoclonal antibody (7E3) against the human platelet GPIIb/IIIa receptor [7E3-F(ab')2] on coronary arterial thrombolysis and reocclusion was studied in a canine preparation of coronary artery thrombosis superimposed on high-grade stenosis. Bolus intravenous injections of rt-PA at a dose of 0.45 mg/kg, repeated at 15 min intervals until reperfusion occurred (maximum of four injections) caused reperfusion in five of seven dogs within 100 min (33 +/- 15 min, mean +/- SD). Reperfusion was rapidly followed (generally within 10 min) by reocclusion and then by periods of cyclical reflow and reocclusion. A single intravenous injection of 7E3-F(ab')2 alone at 0.8 mg/kg caused reperfusion within 100 min in two of six dogs (19 and 37 min) without subsequent reocclusion. Single bolus injections of different amounts (0.1 to 0.8 mg/kg) of 7E3-F(ab')2 were then combined with bolus injections of 0.45 mg/kg of rt-PA. Stable reperfusion without reocclusion was accomplished with 0.8 or 0.6 mg/kg 7E3-F(ab')2 and a single injection of 0.45 mg/kg rt-PA within 6 +/- 3 min (n = 6, p less than .01) and 8 +/- 5 min (n = 5, p less than .02), respectively. None of these animals suffered reocclusion of the coronary artery. Lower doses (0.1 to 0.2 mg/kg) of 7E3-F(ab')2 did not significantly shorten the time to reperfusion and did not prevent reocclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Role of adhesive proteins in platelet tumor interaction in vitro and metastasis formation in vivo.

Platelet-adhesive protein-tumor cell interaction was studied in vitro and in vivo. Monoclonal antibody 10E5, which inhibits binding of fibronectin and von Willebrand factor to the platelet membrane glycoprotein GPIIb-GPIIIa complex, inhibited the binding of mouse CT26 and human HCT8 colon carcinoma cells to platelets by 63-65%, whereas an irrelevant monoclonal antibody, 3B2, had no effect. Monoclonal antibody 6D1, which inhibits binding of von Willebrand factor to GPIb, also had no effect. RGDS, a tetrapeptide that represents the adhesive domain of fibronectin and von Willebrand factor inhibited binding of the tumors to platelets by 64-69%. Monospecific polyclonal antifibronectin antibody inhibited binding by 60-82%; anti-von Willebrand factor antibody inhibited binding by 75-81%. In vivo, polyclonal monospecific anti-mouse von Willebrand factor antibody inhibited pulmonary metastases induced by CT26 tumor cells by 53-64%, B16a amelanotic melanoma cells by 45% and T241 Lewis bladder cells by 46% without induction of thrombocytopenia. Pulmonary metastases with CT26 cells could be inhibited by induction of thrombocytopenia, and reconstituted by infusion of either murine or human platelets. Reconstitution of pulmonary metastases with human platelets could be inhibited 77% by preincubation of human platelets with monoclonal antibody 10E5 before infusion of platelets into mice. Thus, platelets appear to contribute to metastases by their adhesive interaction with tumor cells via the adhesive proteins fibronectin and von Willebrand factor.

Animals↗
PubMed1988

Monoclonal antibody against the platelet glycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs.

Localized thrombosis was produced in the left anterior descending (LAD) coronary artery of open chest dogs by constricting a segment so as to produce greater than 90% stenosis (reducing blood flow to 40 +/- 10% of baseline), and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) at a rate of 15-30 micrograms/kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23 +/- 7 min (mean +/- SD), but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7 +/- 5 min. Intravenous injection of 0.8 mg/kg of the F(ab')2 fragment of a monoclonal antibody (7E3) directed against the platelet GPIIb/IIIa receptor, prevented reocclusion in 10/10 dogs during an observation period of 2 h (P less than 0.001 vs. rt-PA alone). The antibody abolished ADP-induced platelet aggregation and markedly prolonged the bleeding time. Intravenous aspirin or dipyridamole prevented reocclusion for 1 h or more in only 2/7 and 1/6 dogs, respectively. We conclude that the monoclonal antibody is very effective in preventing reocclusion after successful thrombolysis of occluded coronary arteries with rt-PA.

Animals↗
PubMed1988

Structural integrity of the glycoprotein IIb and IIIa genes in Glanzmann thrombasthenia patients from Israel.

Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full-length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.

Blood Platelet Disorders↗
PubMed1987

Plasma glycocalicin. An aid in the classification of thrombocytopenic disorders.

In some patients with thrombocytopenia, it is difficult to determine whether the condition is caused by underproduction of platelets (reduced numbers of megakaryocytes) or an increase in the rate of their destruction (normal or increased numbers of megakaryocytes). A non-invasive test to help distinguish between these two categories of thrombocytopenia would be useful. We related the plasma concentration of glycocalicin, a fragment of the platelet-membrane glycoprotein Ib, to the mechanism of thrombocytopenia by evaluating bone marrow megakaryocyte content and measuring platelet life span. Plasma glycocalicin was measured with a monoclonal antibody to the glycocalicin component of platelet glycoprotein Ib. The mean (+/- SD) plasma concentration of glycocalicin in 34 healthy controls was 87 +/- 20 percent of the level in pooled normal plasma (range, 52 to 127 percent). All of eight patients with aplastic anemia or amegakaryocytic thrombocytopenia confirmed by examining bone marrow (in all patients) and by determining the life span of autologous platelets (in six patients) had glycocalicin levels significantly below the normal range (5 to 27 percent). In contrast, each of 25 patients with thrombocytopenia thought to be caused by a reduction in platelet life span, whose bone marrow contained normal or increased numbers of megakaryocytes, had glycocalicin levels that fell within or above the normal range (48 to 261 percent). There was no overlap of values between the two patient populations. These studies indicate that the measurement of plasma glycocalicin may be a useful adjunct in classifying thrombocytopenic disorders.

Anemia, Aplastic↗