Incorporation of a circulating protein into megakaryocyte and platelet granules.

Abstract

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of a granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into a granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some a granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of a granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.One of the most striking characteristics of platelets is the large variety of proteins that are secreted during the release reaction. Platelets contain counterparts of several plasma proteins (albumin, fibrinogen, and others) in addition to platelet-specific proteins (platelet factor 4 and f3-thromboglobulin). These proteins are localized in a granules, the primary storage organelle for platelet secretory proteins. Predictably, many of these proteins have been shown to be synthesized in megakaryocytes, the bone marrow precursor cell from which platelets are derived. Platelet-specific proteins appear to be synthesized solely by megakaryocytes, whereas proteins such as fibrinogen, coagulation factor V, and von Willebrand factor are synthesized by other cell types in addition to megakaryocytes (1,2).Recently, we discovered that immunoglobulin G is located in platelet a granules and is secreted after platelet activation (3 In Vivo Studies. Male Hartley guinea pigs (300-400 g) were anesthetized with ether, and 75 mg of HRP was dissolved in 1 ml of physiologic saline and injected into the femoral vein. The animals were sacrificed with ether at short (15, 45, and 75 min) and long (7 hr, 24 hr, 48 hr, And 4 days) intervals after the injection. Blood was collected into acid/citrate/dextrose by heart puncture immediately before the guinea pigs were killed. Platelets were counted by use of an automated analyzer (Coulter S + IV). Platelet-rich plasma was obtained by centrifugation of the blood at 100 x g for 10 min at 22°C. Megakaryocytes were isolated from bone marrow according to a described technique (4), e...

Detection of neuropeptide Y and its mRNA in megakaryocytes: enhanced levels in certain autoimmune mice.

“…Immunohistochemical staining of sections from rat bone marrow and NZB mouse spleen with an NPY-specific antiserum revealed large, intensely fluorescent cells, each with a large polymorphic nucleus, identified as megakaryocytes. In situ hybridization confirmed that the NPY-like peptide detected in megakaryocytes of both rat and mouse was synthesized from NPY mRNA present in megakaryocytes and not present as a result of uptake as in the case of horseradish peroxidase (29 They aggregate at the damaged site and release bioactive substances, such as serotonin, that exert a constrictive effect on the vessel wall. NPY, when infused locally into most vascular beds, causes a vasoconstriction with late onset and long duration independent of adrenergic-receptor mechanisms (14,30 aggregation, resulting in a long-lasting vasoconstriction that could supplement the effect caused by (for example) serotonin.…”

Incorporation of intravenously injected albumin, immunoglobulin G, and fibrinogen in guinea pig megakaryocyte granules.

“…In a previous study HRP that had been injected into guinea pigs was seen in coated vesicles in megakaryocytes and subsequently in a-granules (14,15). A similar pathway may be involved in the uptake of plasma proteins by megakaryocytes.…”

“…Biotin-labeled guinea pig albumin (500 mg) was administered intravenously to guinea pigs. Bone marrow megakaryocytes (14) and platelets (18) were isolated as previously described 2 and 24 h after the injection. Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 6 h at 4VC.…”

“…Recently we demonstrated that when the tracer protein horseradish peroxidase (HRP)' (40,000 mol wt) is injected intravenously into guinea pigs, it is rapidly taken up by bone marrow megakaryocytes and incorporated into granules (14,15). Based on this finding, we hypothesized that megakaryocytes may acquire some a-granule proteins from plasma via a similar endocytic pathway.…”

The biogenesis of platelets from megakaryocyte proplatelets

“…Specific proteins associated with platelets, such as vWF and fibrinogen receptors, are synthesized and sent to the megakaryocyte surface, while others are packaged into secretory granules with such factors as vWF, which is loaded into α-granules (17). Still other proteins, such as fibrinogen, are collected from plasma through endocytosis and/or pinocytosis by megakaryocytes and are selectively placed in platelet-specific granules (17,18). Also assembled during megakaryocyte maturation are mitochondria and dense granules, which, like α-granules, derive from Golgi complexes.…”