Makio Murayama

Molecular mechanism of red cell "sickling".

Precision scale models of sickle-cell hemoglobin molecules indicate that the genetic substitution of valine for glutamic acid at the 6th position in the two beta chains allows an intramolecular hydrophobic bond to form. This changes the conformation in such a way as to allow molecular stacking. Optical rotatory dispersion studies and the restilts of suLbjection of Hb S solution to temperature change and to propane are consistent with the presence of such a bond. Examination of sickled erythrocytes in a magnetic field and in polarized light indicates that the Hb S molecules are aligned iiz sitil. Filaments interpreted as hollow cables of six Hb S monofilaments have been demonstrated by electron microscopy.

Ex vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect

Decompression of human platelet-rich plasma (PRP) in siliconized glass or plastic to 380 mm Hg for 3 hours at 38 degrees C produced platelet aggregation independent of pO2. Aggregation also took place when PRP was compressed to 8,000 PSI and then decompressed slowly to one atmosphere (14.7 PSI) without gas bubble formation. Platelets also aggregated when plasma was decompressed hydrodynamically (Bernoulli effect) at room temperature. It was also found that the drugs piracetam (2-oxypyrolidine acetamide) and pentoxifylline (1-(5-oxohexyl)-theobromine) at 0.5 and 1.0 mM prevent thrombocyte aggregation. Implications for mountain sickness are discussed.

Inhibitors of ex vivo aggregation of human platelets induced by decompression, during reduced barometric pressure

It has been shown experimentally ex vivo that human platelet aggregation is induced by decompression (reduced pressure) produced by various means, i.e., reduced barometric pressure, reduced hydrostatic pressure, and reduced hydrodynamic pressure due to Bernoullis principle. We report here that the spontaneous platelet aggregation induced by reduced barometric pressure (253 torr for three hours) is inhibited by 1:10(7) diluted Japanese herbal plant oil (JHP) and also by two of its major constituents, menthone and menthol with the median inhibitory concentration (IC50) in the millimolar range. These drugs gave essentially similar results when collagen and ADP were used as aggregating agents. Inhibitor concentrations were determined by microscopic examination of platelets in wet preparations when the aggregating stimulus was reduced pressure and by optical aggregometry when collagen and ADP were the aggregating agents. Potential usefulness of these compounds in the prevention of decompression syndrome (DCS) and acute mountain sickness (AMS) are discussed.

Compression inhibits aggregation of human platelets under high hydraulic pressure

It has been found in our laboratory that compression induces disaggregation and decompression induces aggregation of human platelets. The experimental design was based on the premise that oppositely charged amino acid residues of fibrinogen are juxtaposed in the specific adhesive site(s). This can be explained by the principle of Le Chateller. Under compression, electrically charged groups gain layers of water molecules which are radially oriented and pulled in closer to the center of the charge, thus making the layer more compact. This increases the thickness of the shield, thus inactivating or disaggregating the platelets. Conversely, decompression drives the water away from the compact shield; water molecules are randomized into the bulk phase; the shield becomes thinner, thus permitting spontaneous aggregation of platelets. It is well known that increasing the concentration of neutral salt can inhibit platelet aggregation. This is due to the increase of ionic strength which attenuates the electric charge and thickens the shield. Molecules having a large electric dipole moment can inhibit platelet aggregation by a similar mechanism. It is generally accepted that fibrinogen is involved in platelet aggregation. However, it is not known whether or not fibrinogen is essential for platelet aggregation inducible by decompression. Here we present data indicating that fibrinogen is essential in decompression-inducible platelet aggregation. We also present experimental data which show that high hydraulic pressure inhibits platelet aggregation, consistent with our premise that electrically charged groups are involved in the initial phase of platelet aggregation.

Sickle Cell Hemoglobin: Molecular Basis Of Sickling Phenomenon Theory And Therap

AbstractThirty years ago, Linus Pauling suggested that sickle cell disease might be due to an abnormal hemoglobin molecule, and I think this was the beginning of molecular medicine. This is what he said about it in his Harvey Lecture69a which he delivered in 1953

Thermal (Or Endothermic) Aggregation of Sickle Cell Hemoglobin (Hb S) During Sickling

The human hemoglobin molecule consists of 4 polypeptide chains, one pair designated the α’s and the other the β’s. Sickle cell hemoglobin (Hb S) differs from the normal by a single amino acid substitution at the 6th position in each β chain. It further appears that there is a hydrophobic pocket in each of the a chains which is complementary to the β-6-Valyl region, allowing deoxygenated sickle cell hemoglobin (Hb S) tetramers to stack by hydrophobic interactions. When oxygenated, the β chains move closer to each other by about 5 angstroms and the stacking crumbles because the goodness of fit is lost, This is unsickling by conformational change. There is another unsickling mechanism which involves an entropy change. In 1957 I reported that Hb S has a negative temperature coefficient of aggregation, i.e., a deoxygenated Hb S solution at 0° gels when warmed to 38° C but liquefies reversibly when replaced in the ice bath. Thus, the sickling phenomenon involves a thermal (or endothermic) aggregation of Hb S. The energy of activation for this reaction (ΔH*) is 17.3 kCal mole-1 and the entropy change amounts to 55 e. u. It appears reasonable to assume that the same entropy change is required to set a molecule of water free from the hydration layer about the hydrophobic residues as is required in the melting of ice.

Pressure influences the rate of fibrin polymerization: decompression accelerates, compression decelerates

We report here that the rate of fibrin polymerization is accelerated by decompression: the rate is essentially doubled by reducing the pressure by one-half (380 torr, 14.7 PSI or 1/2 ATM) and increased by 3.6 times with respect to the control at 25 torr (0.48 PSI or 1/30th ATM). We also report here that the rate of fibrin polymerization is decelerated by compression; at 5.2 X 10(5) torr (10,000 PSI or 680 ATM) polymerization is inhibited and/or reversed.

DIPA (decompression-inducible platelet aggregation) and hemostasis: vascular occlusion and its prevention, in the web of the frog's foot

It is reported here that vascular obstruction is inducible in blood vessels in the web of the frogs foot; the frog had been decompressed for 3 hours at 253 torr (the barometric pressure at the summit of Mt. Everest). The blood flow was reestablished within an hour of recompression at 760 torr. It is reported additionally, that the drug piracetam can block decompression-inducible platelet aggregation (DIPA) and consequently vascular obstruction

Restoration of phagocytosis and oxidative metabolism by Piracetam in failing human neutrophils: A qualitative assessment

By the use of immunobeads, a convenient clinical laboratory test is available which detects, in metabolically stressed leukocytes, failing or absent phagocytosis and/or impaired to absent oxidative metabolic activity. Furthermore, it has been demonstrated that Piracetam, 2-oxo-pyrrolidine acetamide, will restore such compromised neutrophils to normal functional status. In 4 of 19 patients, all with a variety of serious diseases, a range from impaired to total failure of neutrophilic phagocytic and metabolic oxidative activities was detected by the test. Piracetam, as shown by qualitative methods, restored to optimal activity the two impaired neutrophil functions in these 4 patients. Quantitative techniques are available currently to establish the beneficial effect of Piracetam on such defective neutrophils. Piracetam merits additional study to determine its efficacy in enhancement of restorative effects on compromised neutrophils which have been observed. The clinical promise of this investigation offers benefit to some patients now jeopardized by certain stressful diseases in part due to agonal failure of neutrophils.

MOLECULAR ASPECTS OF TACTOID : FORMATION IN MAN AND ANIMALS

In 1940, Sherman reported without interpretation the observation that “under the polarizing microscope characteristic sickle cells show a definite birefringence which disappears after aeration of the cells and the consequent return to the normal discoid form.” In 1950, Harris reported the most significant finding concerning the sickle cell hemoglobin (Hb S) tactoids. He confirmed Sherman’s observation of the birefringence of sickled erythrocyte by examining intact cells under the polarizing microscope after sickling had been induced by deoxygenation. He also noted that normal intact erythrocytes did not become birefringent when treated in the same manner. In deoxygenated Hb S solutions, in the concentration range from 15 to 25 grams per 100 ml, he observed spindle-shaped bodies varying in length from 1 to 15 p in wet preparations under a sealed coverslip. These bodies were birefringent under the polarizing microscope, thereby showing one of the required characteristics of tactoids. Harris noted that these bodies disappeared upon reoxygenation of the hemoglobin solution and reformed when it was deoxygenated again.2 The tactoid is characteristic of an orderly grouping of long, thin, rod-like particles that are parallel and equidistant from each other. Spindle-shaped particles have been observed to form in concentrated solutions of iron oxide and of vanadium pentoxide, and also of several dyes. Bernal and Fankuchena report that tobacco mosaic virus exhibits very well-defined tactoids and they have outlined a theory of their formation. Perutz and Mi tch i~on,~ in 1950, examined deoxygenated suspensions of sickled erythrocytes in 0.1M phosphate buffer at pH 7. The slides were examined under a polarizing microscope with Nicol prisms crossed without compensation; they observed the sickled erythrocytes appeared a deep blue color and were extinguished completely with every 90” rotation of the stage, indicating that sickled cells have the well-known property of parallel extinction. They also studied crystals of normal human deoxygenated hemoglobin and observed a deep blue color and also parallel extinction just like the sickled erythrocytes. These investigators assumed that the plane of the heme is perpendicular to the long axis of the crystal as well as of the sickled erythrocytes. In 1953, Ascenzi and Silverstroni5 studied the optical properties of sickled erythrocytes also with the polarizing microscope and confirmed the findings of