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Biomedical subjects

J M Clemetson

Publications and source records attributed to J M Clemetson.

13 recordsLinked to original sources
PubMed1998

Integrins and cardiovascular disease.

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor alpha IIb beta 3, has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of alpha IIb beta 3 inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.

Arteriosclerosis↗
PubMed1997

Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

Amino Acid Sequence↗
PubMed1996

The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin.

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.

Amino Acid Sequence↗
PubMed1995

Chemokine and chemokine receptor mRNA expression in human platelets.

In order to study the role of platelets in inflammation we constructed a cDNA library from human platelet mRNA. By polymerase chain reaction (PCR) analysis of the library we have shown that platelets express mRNAs for the following chemokines: connective tissue activating peptide-III (CTAP-III), epithelial-derived neutrophil activating factor-78 (ENA-78), RANTES and monocyte chemotactic protein-3 (MCP-3). Platelets also express mRNAs for interleukin 8 receptor A (IL-8RA) and a novel chemokine receptor K5.5. These results suggest that chemokines may not only play an important role in platelet activation but can also influence the nature of the leukocyte infiltrate to sites of inflammation and infection, by the production of multiple chemokines with overlapping specificities.

Base Sequence↗
PubMed1995

Platelet GPIb-V-IX complex. Structure, function, physiology, and pathology.

In the early phase of primary hemostasis, platelets adhere to damaged vessel wall by binding via the platelet glycoprotein (GP) Ib-V-IX complex to von Willebrand factor (vWf) exposed on the subendothelium. The complex is composed of four glycoprotein subunits, GPIb alpha, GPIb beta, GPIX and GPV, each with a variable number of leucine-rich repeats. GPIb alpha and GPIb beta are linked by a disulphide bridge while GPIX and GPV associate noncovalently with the complex. The study of defects in the expression of the GPIb-V-IX complex at the platelet surface leading to pathological disorders, like Bernard-Soulier syndrome (BSS), or in the affinity of platelets for vWf, like pseudo-von Willebrand disease, has helped to delineate the binding site for vWf on GPIb alpha. However, the mechanism by which the complex binds to vWf has not yet been elucidated but it must involve changes in the conformation of the molecules as no interaction between platelets and vWf occurs in the plasma. The GPIb-V-IX complex has a binding site for thrombin on GPIb alpha which participates in the platelet activation by that agonist. GPV is also cleaved by thrombin but the function of this proteolysis is not clear. The platelet response to thrombin is slower and weaker when the thrombin binding site on GPIb alpha is blocked or cleaved or when the GPIb-V-IX complex is not expressed on the platelet surface as in classic BSS. At low doses of thrombin, the rapid activation of the platelets via the seven-transmembrane thrombin receptor is dependent on the presence of the GPIb-V-IX complex.

Endothelium, Vascular↗
PubMed1994

Variant Bernard-Soulier syndrome associated with a homozygous mutation in the leucine-rich domain of glycoprotein IX.

We describe a new variant of Bernard-Soulier syndrome. The patient (W.K.) showed the classic bleeding symptoms together with absence of platelet agglutination to restocetin plus von Willebrand factor, whereas aggregation to ADP, collagen, and arachidonic acid was normal. Platelets were markedly larger than normal and the patient had life-long thrombocytopenia. Surface-labeling of the platelets and two-dimensional gel electrophoresis showed reduced but detectable amounts of glycoprotein (GP) Ib-IX-V present;however, there was markedly less GPIX (2% +/- 1% of normal) than GPIb alpha, Ib beta, or V (7% +/- 2% of normal). This disproportion was confirmed by Western blotting. Sequence analysis was performed after polymerase chain reaction amplification of the coding region of the GPIX and GPI b alpha genes from the patient. A point mutation (A-->G) was found in GPIX converting 45Asn to Ser within the leucine-rich domain. No mutations were found in GPIb alpha. Both alleles of GPIX contained the same defect, which was confirmed by the appearance of a new cleavage site for the restriction enzyme Fnu4HI. This substitution did not affect glycosylation at the neighboring 44Asn as judged by the distribution on two-dimensional gels but did appear to change the conformation of the leucine-rich domain, thus reducing surface expression of the complex. The relationship between GPIb and GPV was not affected, indicating that GPIX does not regulate this. This homozygous mutation in GPIX indicates that, among other possible functions, the leucine-rich domains present on all components of GPIb-IX-V may play a role in the assembly and surface expression of the complex.

Adult↗
PubMed1994

Molecular abnormalities in Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease.

Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. In Glanzmann's thrombasthenia, GPIIb-IIIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional forms have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia.

Antigens, CD↗
PubMed1993

Additional GPI-anchored glycoproteins on human platelets that are absent or deficient in paroxysmal nocturnal haemoglobinuria.

In order to detect novel glycophosphatidylinositol (GPI)-anchored platelet proteins, human platelets were incubated with PI-specific phospholipase C (PI-PLC) and the supernatant was analysed by PAGE and silver-staining for additional protein bands. PI-PLC treatment resulted in the appearance of at least two additional novel GPI-linked glycoproteins (GP), GP500 and GP175, in the supernatant. Their presence on the platelet plasma membrane surface was demonstrated by periodate/[3H]borohydride surface-labelling. Activation of platelets did not enhance the amount of GP500 and GP175 that could be cleaved by PI-PLC. In Triton X-114 phase partitioning of platelet membranes the membrane form of GP175, mfGP175, was in the Triton phase while mfGP500 was found in the water phase. Neither GP500 nor GP175 were present in the supernatant of surface-labelled platelets treated with PI-PLC from 4 patients, diagnosed as having paroxysmal nocturnal haemoglobinuria (PNH), but the supernatant from platelets from healthy volunteers treated the same way contained both.

Blood Platelets↗
PubMed1993

Presence of lipocortins I and IV, but not II and VI, in human platelets.

The present investigation revealed the presence of lipocortins I and IV, but not lipocortins II and VI, in human platelets. Lipocortin I was found in the Triton-soluble fraction of both resting and thrombin-activated platelets and was not covalently bound to skeletal components. Without detergents, when resting platelets were lysed and fractionated in the absence of Ca2+, lipocortin I was found only in the cytosolic fraction, whereas, in the presence of Ca2+, lipocortin I was associated only with the crude particulate and not with the membrane nor the cytosolic fractions.

Annexin A1↗
PubMed1992

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.

Animals↗