Advancements in Neurology:
by Dr. Hiroshi Morooka
Editor’s note: This
paper was originally presented at the 2005 International BWW Conference in
We presented a paper at the 2003 BWW Congress at Antequera: The sympathetic nervous system is especially involved in response to many stress. This sympathetic stimulation to various stress induces activation of procoagulant mechanism on the systematic level. In other words, mental reaction to stress could be responsible for human ageing.
In ischemic process, platelets are activated by agonists such as norepinephrine ( NE ),
epinephrine, arachidonic acid (AA), thromboxane A２(TXA２), serotonin, collagen, adenosine diphosphate (ADP),thrombin and H２O２(reactive oxygen radical).
Foremost among the platelet-derived humoral mediators of tissue injury were the prostaglandins (AA,PGF２α), the endoperoxides (PGH２), and TXA２. TXA２and to a lesser extent, PGF２α and PGH２are potent vasoconstrictors and can reduce blood flow to vital vascular beds. More over, these agents are prothrombotic by virtue of potently stimulating platelet aggregation.
Platelets are the primary source of TXA２. Release of TXA２ aggregate other platelets and stimulates their release of additional TXA２. These platelet propagate process are all considered to be major factors, contributing to direct ischemic injury as occurs in myocardial and cerebral ischemia.
Cellular mechanism of arachidonic acid produces not only TXA２, but also leukotrienes, a group of lipid mediators that initiate, aggravate, or sustain vascular inflammatory disorders.
The cysteinyl leukotrienes ( LTC４,LTD４,and LTE４) constrict vessels and promote vascular permeability. We studied these harmful leukotriens and the role of platelet-leukocyte interaction in hemostasis, thrombosis and atherosclerosis(Fig.1).
Fig.1. Reaction catalyzed by 5-lipoxygenase
Platelet aggregation curve was obtained by the change of light transmission, using NBS HEMA TRACER. 150 patients with cerebral infarction were study participants ( age, 66.96± 9.43years and 71.04± 8.92years; 66 men, 84 women). Platelets rich plasma (PRP) was separated by the centrifugation (800rpm, 15min) of the mixtures, containing 3.8% citric acid / blood (1/9).
Platelet aggregation curves induced by ADP (1.5μM) were divided into 3 types. Type 1: Maximum amplitude of aggregation was less than 40% with dissociated curve from aggregation. Well–treated patients had the type 1 . Type 2: Maximum amplitude was between less than 60% and more than 40%. Type 3: Maximum amplitude was more than 60% with secondary aggregation. Patients with cerebral infarction at acute stage had the type 3.
Patients exposed to stress conditions, including hypertension, smoking, cold temperature, low atmospheric pressure, pain, anxiety, anger, and/or fear, also revealed the type 3 (Fig. 2).
Leukocyte-mediated potentiation of platelet aggregation
Polymorphonuclear (PMN) leukocytes from human blood were obtained by the Hypaque-Ficoll method.１ PMN leukocytes were suspended in the SOLITA®-T No.3
solution, composed of (in w/v %) 0.09NaCl, 0.149KCl, 0.224C３H５NaO３ and 4.3%C6H１２ O６. PMN leukocytes were added to PRP.
Platelet aggregatory response to various agonists in PLATS/PMN (100/1) were more augmented than that in platelet alone(Fig.3).
Fig.3. Leukocytes mediated potentiation of platelet aggregation induced by various agonists such as ADP, collagen, thrombin, AA and phorbol myristate acetate (PMA). 1: Platelets having type 3-aggregation. 2: Platelets having type1-aggregation. Platelet aggregation in PLATS/PMN was potentiated as compared with that in PLATS alone.
Expression of p-selectin in activated platelets
Surface expression of p-selectin is a marker for α-granule secretion in platelet and mediates leukocyte adhesion to activated platelets and endothelial cells.２ Activate platelets produce p-selectin which gathers and activates leukocyte. Leucocyte tethering and rolling has been demonstrated to primarily be mediated by the selectin family of adhesion molecules. Plasma p-selectin was measured using a high-sensitivity enzyme-linked immunosorbent assay (ELISA, Bender Med.Systems).
P-selectin levels in the plasma containing platelets with type 3-aggregation (type 3-platelets) after activation by various agonists were higher than that of control (Fig.4).
Fig.4. Plasma p-selectin levels after platelet activation induced by various agonists.
●: platelets having type 3-aggregation (type 3-platelet),○: platelets having type 1-aggregation (type 1-platelet).
Leukotriene B４production in PMN leukocytes
Leukotrienes were measured using EIA method ( Cayman Chem. Com.).
Leukotriene B４is a potent stimulus for PMN leucocytes, eliciting increases in migration, adherence, degranulation, superoxide production and cytotoxicity.
Levels of leukotriene B４in the plasma containing type 3-platelets mixed with PMN leukocyte after activation by various agonists, were significantly greater than that in Type 3- platelets alone (Fig.5).
Fig.5. Levels of the plasma leukotriene B４after activation of PMN leukocytes. ●: PMN and PLATS,○: PMN alone.
Increased levels of the plasma leukotriene B４, containing type-3 platelets and PMN leukocytes, occurred after activation by various agonists such as ADP, collagen, thrombin, AA or PMA , because platelet-derived AA may serve as a precursor for the 5-lipoxygenase enzyme in the PMN leukocytes.３
Cysteinyl leukotrienes production in interactions between activated platelet and PMN leukocytes..
Leukocytes contain phospholipase A２and 5-lipoxygenase. Thus they can generate LTA４.In contrast, other blood cell, such as erythrocytes and platelets or vascular endothelial cells contain LTA４hydrolase or LTC４synthase, but they lack 5-lipoxygenase and cannot generate their own LTA４. Activated platelet may receive LTA４ from PMN leukocyte ,using transcellular biosynthesis, and produce LTC４.４ LTC４and its metabolites LTD４,LTE４, are potent mediators of vasoconstriction and increase vascular permeability.
The plasma LTB４ and LTE４, containing type3-platelets, revealed high levels as compared with those containing type 1-platelets. Patients with cerebral infarction at acute stage had type-3 platelets (Fig.6).
Fig.6. Enhanced production of leukotrienes in patients with cerebral infarction.
A recent study has shown that C-reactive protein or numbers of leukocytes, a marker of inflammation, was a story predictor of cardiovascular events. It has been postulated for more than a century that infection may be responsible for atherosclerosis.
Chlamydia pneumonia, Cytomegalovirus, Helicobacter pylori, and periodontal infections may be associated with increased risk of carotid atherosclerosis or stroke.5,6,7
These organisms were localized with plaque ulceration and thrombosis. Chronic damaged endothelium may disclose collagen or tissue factors. These substances activate platelet aggregation. Activated leukocyte produce H２O２, leading platelet aggregation.
More over activated leukocytes produce LTA４and LTB４in platelet-leukocyte interaction. Platelet don’t have lipoxigenase, so platelets use LTA４produced by leukocytes and generate LTC４, LTD４and LTE４.These leukotriene may give damage to microcirculation.
Our previous experiments have shown that histamine release is accelerated by free radicals, which may contribute to the increase of macromolecular permeability of the blood brain barrier and to vasogenic edema.８
To treat stroke appropriately, we may use cyclooxygenase inhibitor for TXA２ and also inhibitory drugs for leukotrienes production.
The ways to keep our healthy condition are as follows: 1. We must have mutual relationship and don’t reveal offensive attitude against stress; 2. We should not take much foods , because obesity also cause increase sympathetic stimulation. We should reduce our body weight under the moderate excise; 3. We should take more antioxidant food, containing vitamin E, vitamin C and polyphenols; 4. We have to keep our body away from infection. Antibiotics should be used for suspected infection.
Finally, we should respect individual life and make effort to enhance perpetual peace of all the world with brotherly love.
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H, Sasayama H,
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