Platelets Support the Endothelial Barrier

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Thrombocytopenia in patients is often characterized by petechial hemorrhages due to capillary leakage. Thrombo-cytopenic animals demonstrate gross purpura and alterations in the microvasculature as evidenced by gaps between endothelial cells and leakage of radioactive tracers into the surrounding tissues. The vascular endothelia are thinner and have open pores and membranous diaphragms. Evidence of a dysfunctional endothelial barrier and tissue edema due to thrombocytopenia have been observed in many experimental models such as the frog hindlimb, the thyroid lobe of dogs, the ear of postirradiated rabbits, the thoracic duct lymph of dogs, the coronary microvasculature of rats, and the lungs of sheep. Sheep chronically depleted of platelets with an anti-platelet antibody develop petechial hemorrhages, an increase in the transendothelial clearance of protein in the lungs, and pulmonary edema at a left atrial pressure of 20 mmHg.

Transfusion with platelet-rich plasma reverses these abnormalities. When comparing an isolated, thyroid lobe of a dog perfused with platelet-poor plasma and its counterpart lobe perfused with platelet-rich plasma, the lobe with platelet-poor plasma had gross purpura, endothelial gaps, and increased leakage of protein into the tissue. Transfused platelets labeled with radiosulfate rapidly interact with the vascular endothelium of thrombocytopenic animals and incorporate the label within the endothelium. Interestingly, a common finding is that the number of platelets required to reverse hemorrhage or capillary leakage is much less than that required to correct bleeding times. Thus, platelets in some way nurture the microcirculation.

Three theories have been proposed to explain this endothelial barrier-supporting function of platelets. The first and obvious explanation is that platelets physically block pores or gaps in the vessel wall. The second theory is that platelet components promote endothelial cell growth. The third, and most studied, theory is that platelets continually release a novel humoral factor that decreases endothelial permeability.

The measurement of diffusive permeability across confluent, endothelial cell monolayers developed in the 1980s provided a novel methodology to directly test these three theories. Initial studies addressed the first theory that platelets simply act as plugs between leaky endothelial cells. Washed human platelets, but not paraformaldehyde-fixed platelets, both of which attached sparingly to endothelial cells, reduce the flux of 125I-labeled albumin across endothelial cell monolayers. Subsequently, a number of studies demonstrated that the permeability-decreasing activity of platelets resides in a soluble factor as platelet-conditioned medium (PCM) consistently decreases albumin permeability across endothelial cell monolayers derived from bovine aorta and bovine and human pulmonary arteries and microvessels. Thus, platelets decrease endothelial permeability via the release of a soluble factor and not by plugging of endothelial pores or gaps.

Accepting the notion that this activity of platelets resides in a soluble factor, investigators focused on a number of familiar platelet components such as serotonin, norepineph-rine, and cyclooxygenase metabolites. Proven antagonists or inhibitors of these metabolites did not prevent the decrease in albumin permeability induced by platelets, platelet lysate, or PCM. At the time, norepinephrine (predominately a Pj-adrenergic agonist) was an intriguing possibility because it was known that P-agonists decrease endothelial permeability. This activity of P-agonists, however, favors the P2-subtype, the predominant receptor subtype on vascular endothelial cells.

When my laboratory entered this area of research, adenosine was proposed to be the active platelet factor. We [1] ruled out adenosine as the likely candidate by the following experimental findings. PCM contains micromolar concentrations of AMP, ADP, and ATP, but adenosine is below detectable levels as measured by high-performance liquid chromatography. Although adenosine directly decreases endothelial permeability, inactivation of adenosine with adenosine deaminase, which metabolizes adenosine to inactive inosine, does not block the activity of PCM. Furthermore, the adenosine-receptor antagonist BW-A1433U83 does not block the decrease in endothelial permeability induced by platelets or PCM. A fraction of PCM smaller than 3 kDa that contains micromolar concentrations of AMP and ADP has no activity, whereas a fraction larger than 3 kDa that contains much reduced levels of AMP and ADP significantly reduces permeability. Thus, adenosine and adenine nucleotides are not the primary factors responsible for the platelet activity; instead the activity resides in a fraction of PCM larger than 3 kDa.

As early as 1956, a protein fraction extracted from platelets was reported to reduce capillary permeability in the rat hind limb. Haselton and Alexander [2] and Patil and others [1] demonstrated 35 years later that the active platelet factor is heat-stable, trypsin-sensitive, and >3kDa. Some of these findings point to a protein as the active factor. An obvious difficulty with this notion is how platelets could replenish the store of a protein continuously released into the plasma, considering platelets have a limited ability to synthesize proteins. This criticism prompted us to consider viable alternatives. There were also conflicting pieces of data in the literature. The fact that the active factor is sensitive to trypsin and, in addition, is precipitated in saturated ammonium sulfate implicates a large charged protein, yet the activity is heat stable.

Still assuming that the permeability-decreasing activity of PCM is associated with a protein fraction, Patil et al. [1] performed anion- and cation-exchange chromatography to increase the specific activity of a protein fraction. These experiments provided evidence that the activity resides with a negatively charged protein such as albumin because the active fraction binds to and requires a high salt concentration to elute the activity from an anionic exchange column. Applying PCM to a calibrated Sephacryl S-200 gel filtration column yields three major protein fractions with activity present only in the fraction with an elution volume of albumin. Activity also resides with the albumin immunopreci-pate following removal of albumin from the PCM with an anti-albumin antibody. It was at this time that Alexander and coworkers [3] provided convincing evidence that the active factor is a phospholipid. Their findings pointed to lysophos-phatidic acid (1-acyl-2-hydroxyl-3 phosphoglyceride, LPA).

That LPA might be the active factor was somewhat of a surprise because LPA had been reported to increase the permeability of endothelial cell monolayers derived from brain capillaries. However, the findings of Alexander and coworkers [3] are compelling. Activity is present in the methanol extract of PCM and is eliminated by enzymatic treatment with phospholipase B, which cleaves at the sn-1,2 positions, and with alkaline phosphatase, which cleaves at the phosphomonoester bond. Phospholipase A2, which cleaves at the sn-2 position where a hydroxyl group but no lipid is present in a phospholipid, has no effect. Using a similar protocol, Minnear et al. [4] extracted lipids with methanol from albumin fractions obtained from PCM either by immunoprecipitation with an anti-albumin antibody or by passing the PCM through a Blue-Sepharose column. Successive extractions with methanol yield activity in the methanol extract and loss of activity in the extracted albumin fraction.

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