The phospholipid and fatty acid composition of platelets in patients with primary defects of platelet function

A B S T R A C T The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C.A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity.The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.

Section: Introductionsupporting

confidence: 92%

The effect of phospholipase C on human blood platelets.

Otnaess¹,

Holm²

1976

“…The levels were not measured in patients R. C. and S. C. In the latter study, the normal levels of platelet 5-HT were 126 ±31.6 nmol/10' platelets. A unique phospholipid abnormality, consisting of an increased lecithin: phosphatidyl ethanolamine ratio, was also found in family C but not in the other unrelated patients (29). Through the courtesy of Dr. James A. Wolff, we also studied a 10-yr-old girl (S. M.) of Puerto Rican descent with oculocutaneous albinism (30), on whom some of the findings at age 2 have been previously reported (31).…”

Section: Methods Patientsmentioning

confidence: 98%

Studies of Platelet 5-Hydroxytryptamine (Serotonin) in Storage Pool Disease and Albinism

Weiss¹,

Tschopp²,

Rogers³

et al. 1974

Self Cite

A B S T R A C T Platelets in patients with storage pool disease are markedly deficient in a nonmetabolic (storage) pool of ADP that is important in platelet aggregation. They are also deficient in ATP, although to a lesser degree. In seven patients with this disorder, including one with albinism, platelet 5-hydroxytryptamine (5-HT) levels were reduced in proportion to the reduction in ATP (r = 0.94). Their platelets show diminished capacity to absorb ["C] 5-HT, and the type of defect was similar to that produced in normal platelets by reserpine, a drug known to inhibit the uptake of 5-HT by the platelet dense granules. Storage pool-deficient platelets also converted more [8H] 5-HT to [3H] 5-hydroxyindoleacetic acid than did normal platelets, and the platelets in one of two patients studied contained increased amounts of 5-HT metabolites. The above findings, together with those reported previously, support the conclusion that the capacity of the dense granules (which may be either diminished or functionally abnormal) for storing 5-HT is decreased in storage pool disease; as a result, the 5-HT that enters the platelet may be more exposed to monoamine oxidases present on mitochondrial membranes. This diminished storage capacity (for 5-HT) may also explain why preincubating platelet-rich plasma with 5-HT for 45 min without stirring inhibits subsequent platelet aggregation by 5-HT to a greater degree in patients with storage pool disease than in normal subjects. The latter finding is also consistent with the theory that the aggregation of platelets by 5-HT is mediated by the same receptors on the plasma membrane that are involved in its uptake. The diminished release of platelet-bound ["C]5-HT by collagen that we found in these patients, as well as findings in previous studies, suggests that the release reaction may also be abnormal in storage pool disease.

“…More limited studies on patient T.C., whose platelet aggregation defects are entirely similar to other family members, have disclosed a platelet ATP/ADP ratio of 7.3, and a 5HT value of 213 nmol/ 10" platelets. Other platelet abnormalities in this family include a unique lipid defect (30), increased amounts of glycoprotein IV (31), and a decreased number of a2-adrenergic receptors (8). The clinically affected father of patient D.C. (27) died of documented acute myelogenous leukemia at age 73 yr, and his father, who also had a lifelong bleeding disorder, was also said to have died of leukemia.…”

Section: Methodsmentioning

confidence: 99%

Decreased content and surface expression of alpha-granule membrane protein GMP-140 in one of two types of platelet alpha delta storage pool deficiency.

Lages

Shattil²,

Bainton³

et al. 1991

To determine whether a-granule membranes are present in platelets of patients with storage pool deficiencies of both alpha and dense granules (ab-SPD), we examined the content and surface expression of the a-granule membrane protein GMP-140 in one patient (J.C.) with a severe a-granule deficiency and in three members of a family (family C) with milder a-granule deficiencies. Surface expression of GMP-140 in stimulated platelets, assessed by flow cytometric measurements of the binding of two anti-GMP-140 monoclonal antibodies, was 24-38% of normal values in platelets from patient J.C., vs. 60-95% of normal values in family C. Total platelet content of GMP-140, determined in platelet lysates by antigen-capture ELISA, was 49% of normal in patient J.C., but normal in the members of family C. Platelets of patient J.C. were found to be heterogeneous with respect to GMP-140 content and surface expression by both flow cytometry and immunogold electron microscopy. Approximately 80% of her platelets expressed little or no GMP-140 after stimulation, whereas the remaining 20% expressed normal amounts of GMP-140 and showed extensive immunogold labeling of typical a-granules and clear vacuoles. No such heterogeneity was found in platelets from family C. These findings in the severe ab-SPD patient are in clear contrast to the observations of normal GMP-140 content in the three other ab-SPD patients, and in patients with the gray platelet syndrome, reported previously by others. These results illustrate the phenotypic heterogeneity of a-granule deficiencies in human platelets, and suggest that a defect in granule formation in the megakaryocytes may account for the a-granule defect in at least one form of ab-SPD. (J. Clin. Invest. 1991. 87:919-929.)